RNA G-quadruplex folding is a multi-pathway process driven by conformational entropy

The kinetics of folding is crucial for the function of many regulatory RNAs including RNA G-quadruplexes (rG4s). Here, we characterize the folding pathways of a G-quadruplex from the telomeric repeat-containing RNA by combining all-atom molecular dynamics and coarse-grained simulations with circular dichroism experiments. The quadruplex fold is stabilized by cations and thus, the ion atmosphere forming a double layer surrounding the highly charged quadruplex guides the folding process. To capture the ionic double layer in implicit solvent coarse-grained simulations correctly, we develop a matching procedure based on all-atom simulations in explicit water. The procedure yields quantitative agreement between simulations and experiments as judged by the populations of folded and unfolded states at different salt concentrations and temperatures. Subsequently, we show that coarse-grained simulations with a resolution of three interaction sites per nucleotide are well suited to resolve the folding pathways and their intermediate states. The results reveal that the folding progresses from unpaired chain via hairpin, triplex and double-hairpin constellations to the final folded structure. The two- and three-strand intermediates are stabilized by transient Hoogsteen interactions. Each pathway passes through two on-pathway intermediates. We hypothesize that conformational entropy is a hallmark of rG4 folding. Conformational entropy leads to the observed branched multi-pathway folding process for TERRA25. We corroborate this hypothesis by presenting the free energy landscapes and folding pathways of four rG4 systems with varying loop length. Graphical Abstract

Automated summary

In this study, the folding pathways of a G-quadruplex from the telomeric repeat-containing RNA were characterized using a combination of molecular dynamics simulations and circular dichroism experiments. The presence of ions stabilized the quadruplex fold, and the folding process was guided by the formation of an ion double layer surrounding the charged quadruplex. Coarse-grained simulations accurately captured the ionic double layer using a matching procedure based on all-atom simulations. The results showed that the folding progressed through a series of intermediate states, stabilized by transient Hoogsteen interactions, before reaching the final folded structure. The study also proposed that conformational entropy is a hallmark of rG4 folding, which was supported by the analysis of free energy landscapes and folding pathways of four rG4 systems with varying loop length.