Large-scale conformational analysis explains G-quadruplex topological landscape

Abstract

G-quadruplexes (G4) are four-stranded nucleic acid structures formed within sequences containing repeated guanine tracts separated by 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 shape the conformational landscape of G-quadruplexes. To address these questions, we systematically explored the conformational space of G-quadruplexes using a set of 128 G4-forming DNA sequences with varying loop lengths. Evaluation of nearly 20,000 unique G4 conformations revealed significant foldability differences across 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 conformational landscape and offers valuable insights for the rational design of G4 structures. Author summaryDNA sequences enriched in guanines have the remarkable ability to form helical, four-stranded structures called G-quadruplexes (G4s). These structures have been found across the human genome, where they play a vital role in the regulation of various cellular processes, such as gene expression, replication, or genome maintenance. Moreover, designed G4 structures can be utilized as versatile building blocks in a variety of nanodevices. G4s are characterized by extensive structural diversity, arising from the multiple ways of arranging a DNA strand into a G4. Among the 26 geometrically valid arrangements--called looping topologies--only 14 have been proven experimentally, posing the question of whether the remaining topologies are energetically restricted and, if so, what molecular factors shape the topological landscape of G-quadruplexes. To address this question, in this work, we systematically searched the G4 conformational space for a set of 128 DNA sequences. We used an MD-based de novo folding procedure to evaluate foldability on nearly 20,000 unique G4 conformations. An analysis of the obtained data revealed that the topological landscape of G4s is predominantly restricted by long-distance propeller loops, whose occurrence among topologies is governed by G4s helicity.