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

A one-step and a two-step synthetic pathways were developed for the preparation of non-alkylating and alkylating G4 ligands capable to target selectively G-quadruplex DNA by exploiting click chemistry. Their ability to interact with G-quadruplex secondary structures was evaluated by employing different biophysical assays such as FRET melting and G4 FID assay, and their selectivity towards G4s was tested in the presence of a large excess of duplex DNA competitor. The efficiency of the two-step synthetic approach (functionalization in situ) was validated in vitro in the presence of the biological target and in cells with the development of a visualization technique able to follow G4 ligand distribution in cellular subcompartments. In regard to the alkylating G4 ligands, biochemical assays (gel analyses) were performed for the identification of the ability of these ligands to efficiently alkylate several G4 structures differing in topology and the alkylation reactions thoroughly optimized.

During the first eighteen months of the project, all non-alkylating G4 ligands were successfully synthesized by following both a one-step and the two-step synthetic pathway. The ability of the non alkylating compounds to bind G-quadruplex DNA was analyzed by biophysical assays, in particular by employing FRET melting and G4 FID assay and their selectivity validated in the presence of a large excess of duplex DNA. The two-step synthetic pathway performed in the presence of the G4 target was extensively studied and optimized. Preliminary in cells experiments show the ability of the ligands to penetrate in cells and their cellular distribution was followed by immunochemistry. 14 compounds able to form covalent bonds with G-quadruplex DNA were efficiently synthesized by following exclusively the two-step synthetic approach. Biophysical studies are ongoing. Biochemical assays are performed in the presence of different G4 structures and the alkylation efficiency is under study.

Recognition and resolution of G-quadruplex structures is now of utmost importance in modern molecular genetics as shown by the ever increasing number of publications on this topic. The proposed research project involving new molecular tools able to bind and alkylated G4 structures will enable systematic genome-wide identification of G4 ligand binding sites and, as a consequence, G-rich domains that are likely to fold into quadruplexes in living cells. So far, only a few examples of G4 ligands employed to isolate G4 structures have been reported in literature. However, all of them isolate DNA or RNA G4s from cell extracts not giving information about G4 accessibility in a dynamic system such as a living cell. This is why we propose to exploit alkylating G4 ligands able to recognize and bind G4s in a dynamic system. In addition, by employing these new ligands we will be able to design and develop a new chemical-immunoprecipitation-sequencing methodology (Chem-IP-Seq) to map G4 ligand binding site at single-base resolution. Combining the new Chem-IP-Seq methodology with massive DNA sequencing will enable to gain understanding about G4 generation in cells and their accessibility by G4 ligands making our strategy unprecedented.
At the very end, genome-wide mapping of G4 ligand binding sites in living cells will pave the way for targeted transcriptomic analysis that will allow identifying the effects produced by folding and unfolding of G4s on gene expression.

T. Masson, C. Landras Guetta, E. Laigre, A. Cucchiarini, P. Duchambon, M.-P. Teulade-Fichou, D. Verga*. “BrdU immuno-tagged G-quadruplex ligands: a new ligand-guided immunofluorescence approach for tracking G-quadruplexes in cells”. Submitted Angew. Chemie Int. Ed. (April 2021).

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.