Coexistence of two main folded G-quadruplexes within a single G-rich domain in the EGFR promoter

Abstract

EGFR is an oncogene which codifies for a tyrosine kinase receptor that represents an important target for anticancer therapy. Indeed, several human cancers showed an upregulation of the activity of this protein. The promoter of this gene contains some G-rich domains, thus representing a yet unexplored point of intervention to potentially silence this gene. Here, we explore the conformational equilibria of a 30-nt long sequence located at position −272 (EGFR-272). By merging spectroscopic and electrophoretic analysis performed on the wild-type sequence as well as on a wide panel of related mutants, we were able to prove that in potassium ion containing solution this sequence folds into two main G-quadruplex structures, one parallel and one hybrid. They show comparable thermal stabilities and affinities for the metal ion and, indeed, they are always co-present in solution. The folding process is driven by a hairpin occurring in the domain corresponding to the terminal loop which works as an important stabilizing element for both the identified G-quadruplex arrangements.

Evaluation of the effect of polymorphism on G-quadruplex-ligand interaction by means of spectroscopic and chromatographic techniques

“…It should be stressed that the interpretation of NMR spectra has been a matter of discussion among researchers who defend the formation of either monomeric [26] or dimeric [25] G-quadruplex structures by that sequence. In this regard, the present work highlights the importance of using instrumental techniques to provide complementary information to that obtained through NMR spectroscopy, as shown recently, in the elucidation of the two coexisting monomeric G-quadruplexes, parallel and hybrid, in the promoter region of the EGFT oncogene [40].…”

Duplex‐Guided Refolding into Novel G‐Quadruplex (3+1) Hybrid Conformations

“…[5] Thes sQ oligomer d(GCGTG 3 TCAG 3 TG 3 TG 3 CTCA) with 5'-a nd 3'-overhangs and one three-nucleotide (3-nt) as well as two 1-nt intervening sequences between its four G-tracts is based on at runcated variant of the guanine-rich strand of the nuclease hypersensitive element III 1 in the c-MYC promoter.T he latter has been shown to fold into av ery robust parallel G-quadruplex with three short pro-peller-type loops. [7][8][9] Yet, despite the absence of apparent steric restrictions,acorresponding topology has been elusive to date and not yet unambiguously confirmed, let alone structurally characterized in detail. Notably,s uch afold has been proposed to occur as aminor species for some G4-forming sequences and very recently has also been suggested based on specific cleavage patterns for modified telomeric quadruplexes.…”

“…Notably,s uch afold has been proposed to occur as aminor species for some G4-forming sequences and very recently has also been suggested based on specific cleavage patterns for modified telomeric quadruplexes. [7][8][9] Yet, despite the absence of apparent steric restrictions,acorresponding topology has been elusive to date and not yet unambiguously confirmed, let alone structurally characterized in detail.…”

Duplex‐gesteuerte Umfaltung in neuartige G‐Quadruplex‐(3+1)‐ Hybridkonformationen

“…Ein gezieltes Design von spezifischen Formen würde das Repertoire an G-Quadruplexstrukturen erweitern, die z. [7][8][9] Allerdings blieb trotz des Fehlens offensichtlicher sterischer Einschränkungen eine entsprechende Topologie schwer fassbar und konnte bisher nicht zweifelsfrei nachgewiesen und strukturell charakterisiert werden. Es würde darüber hinaus aber auch helfen, die beim Faltungsprozess wirkenden Kräfte besser zu verstehen.…”

“…Eine Struktur dieser Art wurde bereits als Nebenspezies füre inige Quadruplex-bildende Sequenzen vermutet und kürzlich auch auf Grundlage von Spaltungsmustern bei modifizierten Te lomer-Quadruplexstrukturen vorgeschlagen. [7][8][9] Allerdings blieb trotz des Fehlens offensichtlicher sterischer Einschränkungen eine entsprechende Topologie schwer fassbar und konnte bisher nicht zweifelsfrei nachgewiesen und strukturell charakterisiert werden.…”