Accepted_test

G-quadruplex (G4) structures of DNA in viruses play a crucial role in regulating replication, transcription, and translation. Recently, G4s have garnered attention as a novel approach for developing antiviral strategies. In this study, we focused on genotype 16 of the Human Papillomavirus (HPV), a major cause of cervical cancer, to verify the presence of these potential therapeutic targets. To achieve this, we standardized a sensitive and reliable real-time quantitative PCR (qPCR) stop assay to confirm the in silico predicted G4 structures in the HPV16 genome. Specific primers were designed to amplify putative G4-forming sequences (PQS), targeting specific regions in viral genes and the long control region or promoter (LCR) of HPV16. Additionally, as potassium ions are needed for G4 structures, we determined optimal KCl concentrations and used stabilizing ligands PhenDC3, Pyridostatin (PDS) and BRACO-19 for G4 in vitro formation. The assay successfully confirmed the G4-DNA presence in the early and late genes of HPV16, exhibiting differential reactivity with the mentioned ligands. This underscores the capability of the qPCR stop assay to discern regions with and without DNA-G4 structures, validating its utility as a reliable tool for confirming predictions and investigating these structures. Additionally, we provide a list of potential G4s for future biophysical and functional studies, with potential applications in other HPV genotypes.