Showcasing Different G-Quadruplex Folds of a G-Rich Sequence: Between Rule-Based Prediction and Butterfly Effect

In better understanding the interactions of G-quadruplexes in a cellular or noncellular environment, a reliable sequence-based prediction of their three-dimensional fold would be extremely useful, yet is often limited by their remarkable structural diversity. A G-rich sequence related to a promoter sequence of the PDGFR-β nuclease hypersensitivity element (NHE) comprises a G₃-G₃-G₂-G₄-G₃ pattern of five G-runs with two to four G residues. Although the predominant formation of three-layered canonical G-quadruplexes with uninterrupted G-columns can be expected, minimal base substitutions in a non-G-tract domain were shown to guide folding into either a basket-type antiparallel quadruplex, a parallel-stranded quadruplex with an interrupted G-column, a quadruplex with a V-shaped loop, or a (3+1) hybrid quadruplex. A 3D NMR structure for each of the different folds was determined. Supported by thermodynamic profiling on additional sequence variants, formed topologies were rationalized by the identification and assessment of specific critical interactions of loop and overhang residues, giving valuable insights into their contribution to favor a particular conformer. The variability of such tertiary interactions, together with only small differences in quadruplex free energies, emphasizes current limits for a reliable sequence-dependent prediction of favored topologies from sequences with multiple irregularly positioned G-tracts.