Non-canonical nucleic acids structures in human cells: from molecular interactions to genome-wide mapping

G-quadruplexes (G4s) and i-motifs (iMs) are non-canonical nucleic acid secondary structures that can form in sequences rich in guanine (G) or cytosine (C), respectively. G4s have been extensively characterized both in vitro and in cells, where they have been linked to pivotal biological processes, such as transcription, replication, and genome instability. In contrast, the iM field is much less developed: initially studied in vitro under acidic conditions, their formation in cells is still under debate. In the present thesis, we evaluated the biology of quadruplexes from two different perspectives: molecular investigation of specific G4-interacting proteins and genome-wide mapping of both G4s and iMs. We explored the presence of specific proteins capable of interacting with a G4 located in the promoter of CDK4, the second most highly amplified gene in a rare type of poorly curable human liposarcoma. Using pull-down LC-MS assays, we identified Ku70 as CDK4-G4 interacting protein. Although Ku70 function is mainly documented in the DNA repair process, other activities have been associated with it, such as transcription modulation and helicase activity. Since these functions may be related to the presence of G4 in the CDK4 promoter, we investigated the interaction of Ku70 with CDK4-G4 in detail. We confirmed the ability of Ku70 to bind CDK4-G4 in vitro and showed that this interaction also occurs in cells. We extended the analysis all cellular G4s performing immunofluorescence-based assays in liposarcoma cells and demonstrated the ability of Ku70 to bind different G4s in cells. To identify the genomic regions where this interaction occurs, we employed CUT&Tag using anti-Ku70 and anti-G4 antibodies, which data analysis is still under investigation. These results represent one of the first attempts to ascribe a novel G4-related function to Ku70 in the chromatin context, beyond DNA repair. In addition, we extended the genome-wide investigation of non-canonical secondary structures to iMs. We performed CUT&Tag using the anti-iM antibody and showed for the first time that iMs form within the human genome in living cells. We mapped iMs in tumoral and non-tumoral cell lines and recovered sequences that were confirmed to fold into iMs in vitro. We found that iMs in cells are mainly located at actively transcribing gene promoters, in open chromatin regions, they overlap with R-loops, and their abundance and distribution are specific to each cell type. By simultaneously mapping G4s, and comparing the results with iMs, we proved that the two structures can form in independent regions; however, when both iMs and G4s are present in the same genomic tract, their formation is enhanced. iMs and G4s were mainly found at genes with low and high transcription rates, respectively. Our findings support the in cells formation of iM structures and provide new insights into their interplay with G4s as new regulatory elements in the human genome. Since the G4-/iM-landscape is influenced by different cellular environments, we further investigated G4 formation in the human genome in the context of infection-induced cellular stress. Using CUT&Tag, we provided the first host G4-landscape mapping during the first hours of infection by two evolutionarily distant viruses: HSV-1 and SARS-CoV-2. We demonstrated that the cellular G4-landscape is highly responsive to infection, particularly at the onset of genome replication. By analyzing the infection-induced changes, we showed that not all the infection-induced changes in the host G4-landscape differ depending on the virus infecting the cell. We identified a common host response to viral infection characterized by G4 enriched genes pathways. These results support the identification of a defined host response that is independent of the human cell type and the virus infecting the cell, and provide the basis for the discovery of innovative antiviral compounds targeting the G4s that sustain viral infections.