How loop lengths shape topological preferences of two-tetrad G-quadruplexes

Smallest G-quadruplex (G4) structures comprise only two stacking G-tetrads. DNA sequences capable of forming such folds are extremely abundant in the human genome and a growing body of evidence shows that even these simplest G4 motifs play important roles in both gene regulation and in numerous pathological processes. However, our understanding of the sequence–structure relationship governing two-tetrad G4 formation remains limited. In the current work, we provide a systematic analysis of how loop length shapes the structural preferences of two-tetrad G4s by experimentally characterizing G4s formed by all 64 DNA sequences of the type 5′-GGTx=1–4GGTy=1–4GGTz=1–4GG-3′. Using a combination of biophysical methods, we were able to distinguish two-tetrad G4s from higher-order G4 assemblies and define several features in experimental data that are characteristic of this kind of structures. Through 2D NMR (nuclear magnetic resonance) analysis, we assigned specific loop topologies for 17 two-tetrad folds, identifying the +l+l+l (antiparallel-chair) topology as the most prevalent, followed by −ld+l (antiparallel-basket), with fewer examples of d+pd (antiparallel-basket) and −l−l−l (antiparallel-chair) G4s. Taken together, the gathered dataset delineates regions of sequence space occupied by G4 folds of different molecularities and loop topologies, revealing the length of the central loop as the main determinant of two-tetrad G4s’ structural preferences.