Structural Insights into Spare-Tire DNA G-Quadruplex from the Human VEGF Promoter

The vascular endothelial growth factor (VEGF) promoter region, which is involved in cancer progression, con-tains guanine-rich sequences capable of forming G-quadruplex (G4) structures. G4s play a critical role in transcriptional regulation and genomic stability and exhibit high structural polymorphism. The major VEGF G4 adopts a parallel topology involving the first four of five G-tracts (VEGF1234), while a potential "spare-tire" mechanism suggests the formation of VEGF1245 in response to oxidative damage. Here, we characterize this alternative G4 (VEGF1245), formed by excluding the third G-tract, using circular dichroism (CD) and nu-clear magnetic resonance (NMR) spectroscopy. Structural analysis reveals that VEGF1245 folds in a hybrid conformation. Different to other five tract containing G4s, for which various stand topologies can rapidly interconvert, VEGF1245 remains thermodynamically metastable and does not refold spontaneously into VEGF1234 at physiological temperatures. Further trapping of the VEGF1245 conformation by a photolabile protecting group and its in-situ release documents that the transition to VEGF1234 requires elevated tem-peratures, implicating kinetic barriers in the refolding process and the delineation of VEGF1245 as prominent metastable conformation. Our findings provide new insights into transcriptional regulation and DNA repair for the cancer-related VEGF-G4.