Solution structure of the major G-quadruplex formed in the human VEGF promoter in K+: insights into loop interactions of the parallel G-quadruplexes

Abstract

Vascular endothelial growth factor (VEGF) proximal promoter region contains a poly G/C-rich element that is essential for basal and inducible VEGF expression. The guanine-rich strand on this tract has been shown to form the DNA G-quadruplex structure, whose stabilization by small molecules can suppress VEGF expression. We report here the nuclear magnetic resonance structure of the major intramolecular G-quadruplex formed in this region in K+ solution using the 22mer VEGF promoter sequence with G-to-T mutations of two loop residues. Our results have unambiguously demonstrated that the major G-quadruplex formed in the VEGF promoter in K+ solution is a parallel-stranded structure with a 1:4:1 loop-size arrangement. A unique capping structure was shown to form in this 1:4:1 G-quadruplex. Parallel-stranded G-quadruplexes are commonly found in the human promoter sequences. The nuclear magnetic resonance structure of the major VEGF G-quadruplex shows that the 4-nt middle loop plays a central role for the specific capping structures and in stabilizing the most favored folding pattern. It is thus suggested that each parallel G-quadruplex likely adopts unique capping and loop structures by the specific middle loops and flanking segments, which together determine the overall structure and specific recognition sites of small molecules or proteins.LAY SUMMARY: The human VEGF is a key regulator of angiogenesis and plays an important role in tumor survival, growth and metastasis. VEGF overexpression is frequently found in a wide range of human tumors; the VEGF pathway has become an attractive target for cancer therapeutics. DNA G-quadruplexes have been shown to form in the proximal promoter region of VEGF and are amenable to small molecule drug targeting for VEGF suppression. The detailed molecular structure of the major VEGF promoter G-quadruplex reported here will provide an important basis for structure-based rational development of small molecule drugs targeting the VEGF G-quadruplex for gene suppression.

Ionic Effects on VEGF G-Quadruplex Stability

“…44,45 The CD-detected parallel structure of The Journal of Physical Chemistry B Article the G-quadruplex adopted by Pu22-T12T13 in the presence of potassium ions is consistent with the NMR solution structure. 35 Figure 1b shows the folding profile of Pu22-T12T13 as monitored by the K + dependence of the molar ellipticity at 264 nm. It can be seen from Figure 1b that the Pu22-T12T13 Gquadruplex formation is complete above ∼2 mM KCl.…”

“…In potassium solution, Pu22-T12T13 has been shown to adopt a single parallel-stranded G-quadruplex conformation with a 1:4:1 loop-size arrangement. 35 In the Pu22-T12T13 Gquadruplex, the four-nucleotide middle loop stretches over the terminal quartet, forming a specific caping structure involving the two flanking segments. 35 This structural feature of Pu22-T12T13 is in contrast to our previously studied c-MYC G-quadruplex in which the two-nucleotide middle loop remains in the groove.…”

Oligomer formation and G-quadruplex binding by purified murine Rif1 protein, a key organizer of higher-order chromatin architecture

“…Interaction of muRif1 with characteristic subsets of G4 DNA ~ There are several well known G4 oligonucleotides that have been structurally characterized (38)(39)(40)(41)(42)(43)(44)(45)(46). We used ten of them for EMSA with purified muRif1 (Supplementary Fig.…”

Oxidative DNA damage is epigenetic by regulating gene transcription via base excision repair

“…1B) (10). Our previous work mapped the most reactive guanine (G) bases in VEGF toward oxidation, leading to OG and other secondary oxidation products (11).…”