Genome-wide mapping of G4-ligand binding sites at single-base resolution – NEMESES

Oligonucleotides containing runs of three or four adjacent guanines may spontaneously arrange into four-stranded DNA supramolecular structures called G-quadruplexes (G4s). These non-canonical structures are likely to form in G-rich regions throughout the genome suggesting possible functional roles in key biological processes. Currently, G4s are thought to represent a possible third level of genetic regulation and cumulative data suggest G4s to be linked with human diseases. Therefore, they have become objects of intense study. However, the dynamic nature of these secondary structures makes their identification in living cells extremely difficult and for this reason it is a challenge to definitely establish their biological relevance.
The knowledge of G4 structures about their location in the genome and their dynamics in cells are important aspects for further exploring G4 functions and its significance in both biology and medicine.
The aim of this project is to develop and apply techniques that overcome the current limitations for mapping G4s in cells and to identify loci-specific G4 structures at the genome-wide level. This project will allow to develop DNA structure/function elucidation techniques that couple potent small synthetic molecules (G4-ligands) with high-throughput DNA sequencing to directly and globally map G4 structures in cells and reveal their functional and regulatory roles in mammalian cells.
These objectives will be achieved by the construction of specifically tagged G4-selective non-alkylating and alkylating molecules that will enable systematic genome-wide identification of G4 ligand binding sites and, as a consequence, identification of G-rich domains that are likely to fold into quadruplexes in living cells. This new ligands will allow the development of a new chemical-immunoprecipitation-sequencing methodology (Chem-IP-Seq) to map G4 ligand binding site at single-base resolution.
Our strategy is expected to provide answers to the current questions concerning the consensus sequences forming G-quadruplexes in vivo, thereby contributing to a deeper understanding of the G4 accessibility, dynamic, and regulatory roles; in particular, their connection to mutational processes associated with diseases. Unprecedently, the mapping of G4 ligand binding sites at single-base resolution will allow to connect cellular response with the exact binding site of the ligand. In fine overarching aim of this work is to validate G4s as therapeutic targets and pave the way for targeted transcriptomic analysis that will allow identifying the biological responses induced by folding and unfolding of G4s on gene expression.