In-Gel Probing Polymorphic Structures of G-Quadruplexes Derived from c-Myc Promoter

Abstract

Abstract Some G-quadruplex (G4) forming sequences are able to form multimers under physiological conditions, which limits the structure-activity study of G4s. Here we report an in-gel visual method to probe the conformational polymorphism of the derivatives of a parallel G4-forming sequence, c-Myc by flanking with different A/T bases and typical intermolecular G4-forming sequences. Fluorescence and circular dichroism (CD) spectra showed that the interaction of c-Myc derivatives with Thioflavin T (ThT) caused different fluorescence enhancements of ThT, and intermolecular parallel G4s caused higher fluorescence enhancement of ThT than that of intramolecular parallel and intermolecular antiparallel G4s. These DNA sequences were separated on native gels and stained by ThT for G4 and then by Stains-all (dye) for all DNA structures. Most of the sequences adopted parallel G4 multimers and intramolecular G4s in the presence of K+, meanwhile only a part of them formed G4 multimers and intramolecular structures in the presence of Na+. The optical density (OD) ratio of DNA bands stained by ThT and Stains-all were derived by ImageJ, and the ratios of G4 multimers were higher than 1.0, intramolecular parallel G4s were about 0.5, and non-G4s were near 0. Furthermore, the flanking nucleotides were favorable to the formation of multimers of c-Myc in K+ solutions, especially those flanking with T; while in Na+ solutions, only those sequences flanking with two or more A preferred to form G4 multimers. Our results suggest that the current strategy holds the potential for polymorphic study of G4s.