Detection of a G-Quadruplex as a Regulatory Element in <i>Thymidylate Synthase</i> for Gene Silencing Using Polypurine Reverse Hoogsteen Hairpins

• Main Authors: Eva Aubets, Alex J. Félix, Miguel Garavís, Laura Reyes, Anna Aviñó, Ramón Eritja, Carlos J. Ciudad, Véronique Noé
• Format: Article
• Language: English
• Published: MDPI AG 2020-07-01
• Series: International Journal of Molecular Sciences
• Subjects: G-quadruplex; PPRH; thymidylate synthase; anti-tumor therapy; 5-fluorouracil; gene silencing
• Online Access: https://www.mdpi.com/1422-0067/21/14/5028
• ISSN: 1661-6596; 1422-0067

Thymidylate synthase (TYMS) enzyme is an anti-cancer target given its role in DNA biosynthesis. TYMS inhibitors (e.g., 5-Fluorouracil) can lead to drug resistance through an autoregulatory mechanism of TYMS that causes its overexpression. Since G-quadruplexes (G4) can modulate gene expression, we searched for putative G4 forming sequences (G4FS) in the <i>TYMS</i> gene that could be targeted using polypurine reverse Hoogsteen hairpins (PPRH). G4 structures in the <i>TYMS</i> gene were detected using the quadruplex forming G-rich sequences mapper and confirmed through spectroscopic approaches such as circular dichroism and NMR using synthetic oligonucleotides. Interactions between G4FS and TYMS protein or G4FS and a PPRH targeting this sequence (HpTYMS-G4-T) were studied by EMSA and thioflavin T staining. We identified a G4FS in the 5’UTR of the <i>TYMS</i> gene in both DNA and RNA capable of interacting with TYMS protein. The PPRH binds to its corresponding target dsDNA, promoting G4 formation. In cancer cells, HpTYMG-G4-T decreased TYMS mRNA and protein levels, leading to cell death, and showed a synergic effect when combined with 5-fluorouracil. These results reveal the presence of a G4 motif in the <i>TYMS </i>gene, probably involved in the autoregulation of TYMS expression, and the therapeutic potential of a PPRH targeted to the G4FS.