Can intrinsic loop energetics predict G-Quadruplex topology?

Naturally occurring DNA G-quadruplexes (G4s) regulate many cellular processes, such as gene expression and replication, whereas designed G4s serve as building blocks for controllable nanodevices. Composed of four G-tracts and three loops, G4s exhibit high structural polymorphism, mainly associated with different geometries adopted by the loops. Understanding the biological role of G4s and facilitating their targeted design require detailed knowledge of their folding preferences, which likely originate from the stability of individual loops. However, sequence-dependent preferences for loop geometries and their effects on the overall G4 conformational landscape remain unclear. Here, we used molecular dynamics simulations to systematically evaluate folding free energies of all five standard G4 loop geometries across four loop lengths. Our results reveal that loop-geometry preferences strongly depend on the loop spatial span and, with increasing length, shift from low-span toward large-span geometries. Crucially, we discovered that the overall G4 fold is primarily dictated by internal loop stabilities in three-tetrad G4s but not in two-tetrad ones, where inter-loop interactions emerge as vital stability determinants. Moreover, internal loop stabilities explain the exceptional structural diversity of G4s with three-nucleotide loops reported by experimental studies. Finally, we show that loop-geometry preferences arise from an interplay between the electrostatic repulsion of phosphate groups and loop overstretching.