Na⁺-concentration dependent conformational switch of oncogene RET G-quadruplex DNA in solution

Proto-oncogene RET is overexpressed in many cancers, and its expression level is positively related to the size and malignancy of the tumors. Effective inhibition of its overexpression can be used to potentially treat cancers. A guanine-rich GC-boxes (I-V) sequence in its promoter region folds into noncanonical G-quadruplex (G4) DNA structures, negatively regulating its expression by interactions with small molecules. Previously, we reported that RET20T in GC-boxes II-V formed parallel G4 only at low concentrations of K⁺ solutions, and that RET-21mer in GC-boxes I-IV folded into different mixed parallel / antiparallel G4s in 50 mM K⁺ or 100 mM Na⁺ solutions. These data implied high complexity in producing G4 topology by cation type. Here, mainly by nuclear magnetic resonance (NMR), we demonstrated that RET20T formed slightly different, but rarely reported inter-convertible mixed parallel / antiparallel G4s dependent on Na⁺ concentration. Unclassical base-pairs G³•G⁶ and G¹⁴•T²⁰ and ternary plane G³•C⁵•G⁶ stabilized these two RET20T G4 structures. These structural evidences enhanced our understanding that generation of a unimolecular G4 structure could be significantly affected by cation concentration and type in buffer, and the oligo sequence. Conformational switch of G4s should be noted before anti-cancer drug screening in future.