The folding of DNA molecule into non-canonical secondary structures has been shown to be implicated in many important biological processes which regulate cell proliferation and proteins expression. In particular one of these peculiar secondary structures, called G-quadruplex (G4), has been shown to potentially impair cancer development. G4 occurs along DNA sequences rich of consecutive guanines which can fold through Hoostein pairs by forming stacked planes of guanines tetrads. This conformation prevalently forms along the termini of chromosomes (telomeres) but also along the promoter sites of several oncogenes directly involved in many cancers. The G4 formation leads to an hindrance on DNA molecule which hinder the telomere elongation and transcription process. The result is a switching off of these mechanisms which are directly involved in cancer progression. Several factors can influence the G4 equilibria for example, saline conditions, temperature, pH, the binding with specific proteins as well as the presence of dehydrating cosolutes. Additionally, the overall structural feature of the G4 is strictly dependent upon the DNA sequence. As a results, different G4 can be identified inside the cells.
In this project, we focused on the conformational study of the promotorial regions of EGFR and BRAF oncogenes since, on these sites the existence of G4 putative forming regions was found. In particular, the sequences at positions -272, -37 of EGFR and -176 of BRAF from the transcription start site were analyzed. Indeed, no previous literature data were reported about the structural equilibria in solution of these sequences. We found that our tested sequences are actually able to fold into G4 by setting the most proper experimental conditions and also close to the intracellular physiological environment (KCl 150 mM, pH 7.5).
However, oncogenes are double stranded sequences and the folding of the complementary cytosine rich strand into i-motif (iM) can be involved in the switching off of gene transcription. Although, so far, no physiological evidence has been observed for i-motif conformation, here, we aimed to investigate also the cytosine rich strand conformation, to assess if this folding in the case of our sequences is compatible with the physiological conditions and if it can synergically works with the G4 to destabilize the double strand. Our data showed that in physiological condition the preferential form is represented by the double strand . However, some selected ligands showed to shift the DNA B-form toward the non canonical conformation. Indeed, here we implemented our work with the screening of two libraries of compounds in order to find a selective and efficient binder. We carried on the binding study of anthraquinones and naphthalene diimides derivatives, known to have the chemical features of efficient G4 binders. These ligands were first tested on different G4 templates, known to be validated models for G4 binding study, and their efficiency on G4 has been compared with the double strand. The most G4 selective derivatives were than investigated towards our oncogenic G4s. Although more work is required to identify a lead compound, we were able to demonstrate how the use of asymmetrical substitution pattern on a aromatic core can implement the selectivity among different G4s.
Finally, in order to map the occurrence of G4 conformation in vivo, we set up a novel technique which consists in an in vivo footprinting protocol. This work, performed at University of Mississippi, Oxford, MS (USA), under the supervision of Dr Tracy A. Brooks, should provide novel insight on the G4 formation in the cells according to their physiological and environmental condition
