Identification of Ligand-Responsive RNA G-Quadruplexes in the 3' UTRs of Dengue Virus Serotypes

Dengue virus (DENV), which comprises four antigenically distinct serotypes (DENV-1 to DENV-4), remains a major global public health concern and continues to expand geographically; however, the structural features of the viral genome remain incompletely understood. Although G-quadruplexes (G4s) have previously been reported in coding regions of DENV, their presence within the 3' untranslated region (3' UTR) has not been experimentally characterized. Here, we focused on selected guanine-rich motifs within the 3' UTRs of DENV-1 to DENV-4 and investigated their ability to form RNA G4 structures. Using bioinformatic analysis, we identified comparable G-rich regions in the 3' UTRs of the four serotypes, with serotype-dependent differences in conservation. We then examined the propensity of the selected putative quadruplex-forming sequences (PQSs) to adopt G4 structures using circular dichroism spectroscopy, UV melting analysis, ¹H NMR spectroscopy, ligand-binding analysis, and reverse transcription stop (RT-stop) assays. Our results provided in vitro evidence that the 3' UTR oligonucleotides from DENV-1 to DENV-4 are capable of forming ligand-responsive G4 structures, with serotype-dependent differences in conservation, stability, and conformational homogeneity. In addition, reverse transcription (RT)-stop analysis revealed ligand-dependent arrest at the corresponding PQS sites in the presence of the G4 ligand 6OTD, which stabilizes G4 structures. These findings suggest the DENV 3' UTR as an additional source of ligand-responsive RNA G4-forming elements and support future studies on their possible roles in DENV RNA regulation.