Large-Scale Conformational Analysis Explains G-Quadruplex Topological Landscape

G-quadruplexes (G4) are four-stranded nucleic acid structures formed within sequences containing repeated guanine tracts separated by intervening loop regions. Abundant in the human genome, they play crucial roles in transcription regulation and genome maintenance. Although theoretically capable to adopt 26 different folding topologies─primarily differing in loop arrangements─only 14 of these have been observed experimentally. This raises fundamental questions about whether the remaining topologies are energetically inaccessible and what molecular factors determine the folding patterns of G-quadruplexes. To address these questions, we systematically explored the conformational space of G-quadruplexes using a set of 128 DNA sequences capable of forming two- and three-tetrad structures with varying loop lengths. We conducted foldability evaluations of nearly 20,000 unique G4 conformations obtained through an in silico folding procedure. Our analysis revealed significant differences in foldability among the 26 theoretical topologies. Crucially, we demonstrated that the presence of long-distance propeller loops in 12 of these topologies imposes strict loop length constraints, hindering their formation, especially in sequences with shorter loops. Additionally, we found that the occurrence of long-distance propeller loops is governed by G4 helicity, resulting in opposite folding preferences in right-handed and left-handed G4s. By providing geometric explanation for G4 folding patterns, our study advances the understanding of the G-quadruplex topological landscape and offers valuable insights for the rational design of G4 structures.