DNA structural polymorphism in the proximal promoter of the vascular endothelial growth factor gene

3189 Vascular endothelial growth factor (VEGF) plays an important role in tumor angiogenesis. A polyguanine/polycytosine (polyG/polyC) tract in the proximal promoter of the VEGF gene is essential for its promoter activity. Because this polyG/polyC tract is very dynamic in nature, it is able to unwind into single-stranded DNA and adopt non-B-DNA conformations, which potentially play a role in VEGF transcriptional regulation. In this study, we characterized the secondary structures that are formed in the guanine-rich (G-rich) and cytosine-rich (C-rich) strands of this tract using various biochemical and biophysical techniques. It was revealed that, in the presence of 100 mM KCl, the G-rich strand is able to adopt two parallel G-quadruplex loop isomers containing three G-tetrads with three double-chain-reversal loops of either 1:4:1 or 1:2:3. Our CD melting experiment and polymerase stop assay suggested that the G-quadruplex containing 1:4:1 double-chain-reversal loops is the more thermodynamically stable conformation that the G-rich strand readily adopts. This provides strong evidence that the sequences and sizes of the loops are critical for the formation and stability of G-quadruplexes. Our molecular model showed that the guanines at the 5’-end and the 3’-end of the G-quadruplex provide a stable capping structure. The complementary C-rich strand is able to form an intramolecular i-motif structure at slightly acidic pH, which involves six C-C+ base pairs and three loops of 2:3:2. One guanine and cytosine in one of the loops stabilize the i-motif by stacking interactions. Taken together, our results demonstrate that the G-quadruplex and i-motif structures are able to form in the G-rich and C-rich strands respectively of the polyG/polyC tract in the VEGF proximal promoter under conditions that favor the transition of B-DNA to non-B-DNA conformations. These unique secondary structures may be novel targets for anti-angiogenesis therapy in cancer.