DNA G-quadruplexes (G4-DNA) are DNA secondary structures formed in specific G-rich sequences. The G4-DNA forming sequences are found in regions of the genome with biological significance, such as telomeres or oncogene-promoters, for example. G4-DNA has recently emerged as a new promising class of molecular targets in oncology, as transcriptional repression of oncogenes through stabilization of these structures could be a novel anticancer strategy. Many gene promoter G-quadruplexes have structural characteristics that make them druggable, and their inherent structural polymorphism resulting in structural diversity indicates that high degree of selectivity might be achieved. Number of small molecular weight ligands that bind and stabilize telomeric G4 structures in vitro have shown remarkable anticancer activity in tumor xenograft models and have entered clinical trials. However, in vivo, all the ligands failed to selectively target telomeric G4-DNA relative to other G4-forming regions in the genome. Recent studies strongly suggest that lack of information on both high-resolution structures that these G4 forming sequences adopt under in vivo conditions and on degeneracy of structural space among various G4-DNA in the genome are the key limitations for rational development of ligands selectively targeting particular G4-forming regions. In this project, using c-MYC and hTERT oncogene-promoters as paradigm, we will develop a pipeline (based on state-of-the-art approach of in-cell NMR spectroscopy) for in vivo characterization of physiologically relevant conformational equilibriums of G4-DNA in oncogene-promoters. Moreover, the in-cell NMR will be used to assess selectivity of prototypic G4-binding ligands to particular G4-conformational type inside living cells. The proposed research is expected to provide rational basis leading towards selective targeting of DNA G-quadruplexes by small molecular weight ligands within the human genome.

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