Elucidating the solution structure of the monomolecular <i>BCL2</i> RNA G-quadruplex: a new robust NMR assignment approach

5' untranslated regions (UTRs) of mRNA commonly feature G-quadruplexes (G4s), crucial for translational regulation and promising as drug targets to modulate gene expression. While NMR spectroscopy is well-suited for studying these motifs' structure and dynamics, their guanine-rich nature complicates resonance assignment due to high signal overlap. Exploiting the inherent rigidity of G4 cores, we developed a universally applicable assignment strategy for uniformly isotopically enriched G4 structures, relying solely on through-bond correlations to establish the G-tetrads. Applying this approach, we resolved the solution structures of two triple mutants of the RNA G4 in the 5' UTR of the human <i>BCL2</i> proto-oncogene, one of the first natural monomolecular RNA G4 structures available to date. Comparative analysis with other RNA and DNA G4s reveals their notably compact and well-defined cores. Moreover, the sugar pucker geometries of the tetrad guanines are far less stringent than previously assumed, adeptly accommodating specific structural features. This contrasts with the canonical base pairing in RNA and DNA, in which the sugar pucker dictates the type of the double-helical structure. The strategy presented provides a direct path to uncovering G4 structural intricacies, advancing our grasp of their biological roles, and paving the way for RNA-targeted therapeutics.