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

Area of Science:

• Molecular Biology
• Biophysics
• Structural Biology

Background:

• 5' untranslated regions (UTRs) of mRNA contain G-quadruplexes (G4s) that regulate translation.
• G-quadruplexes are promising drug targets for modulating gene expression.
• NMR spectroscopy is ideal for studying G4 structure and dynamics, but signal overlap complicates resonance assignment.

Purpose of the Study:

• To develop a universal NMR assignment strategy for G-quadruplex structures.
• To determine the solution structures of RNA G4s in the 5' UTR of the human BCL2 proto-oncogene.
• To compare the structural features of RNA G4s with DNA G4s.

Main Methods:

• Developed a novel NMR assignment strategy exploiting G4 core rigidity and through-bond correlations.
• Applied the strategy to uniformly isotopically enriched G4 structures.
• Resolved the solution structures of two triple mutants of the BCL2 RNA G4.

Main Results:

• Successfully assigned resonances and determined the solution structures of two BCL2 RNA G4 mutants.
• Identified notably compact and well-defined cores in these RNA G4s compared to other G4s.
• Demonstrated that sugar pucker geometries in tetrad guanines are more flexible than previously assumed.

Conclusions:

• The new NMR strategy enables detailed structural analysis of G-quadruplexes.
• RNA G4s possess distinct structural properties, including flexible sugar pucker geometries.
• This work advances understanding of G4 biological roles and supports RNA-targeted therapeutic development.