NMR and molecular dynamics demonstrate the RNA internal loop GAGU is dynamic and adjacent basepairs determine conformational preference.

The conformational variability of RNA duplexes with internal loop 5'GAGU/3'UGAG was investigated by nuclear magnetic resonance (NMR) spectroscopy and all-atom molecular dynamics (MD) simulations. It was previously found that the CG-flanked internal loop in 5'GACGAGUGUCA/3'ACUGUGAGCAG existed in a major conformation (I) characterized by U7 and U7∗ bulging into solution, A5 and A5∗ stacking, and G4-G6∗ and G6-G4∗ basepairs closing the loop. A minor conformation also existed with G-U pairs with a bifurcated hydrogen bond and A-G pairs with a single hydrogen bond (II). A maximum hydrogen bonding structure with wobble G-U pairs and two-hydrogen-bond A-G pairs (III) was not observed by NMR or MD. Here, flanking pairs A-U, U-A, and G-C were substituted for the C-G basepair adjacent to the GAGU internal loop. NMR demonstrated changes in conformational preference depending on the identity of the flanking pair. Clustering of MD structures for C-G- and U-A-flanked duplexes showed transitions between conformations II and III with a greater fraction of the structures in conformation II, although the simulations started in conformation III. Additionally, U-A-flanked simulations contained 25% of structures in a state between conformations II and III. A-U- and G-C-flanked structures were in a single cluster whose centroid was conformation II. MD showed a dominance of structure II over III, in agreement with NMR for C-G closure, but in disagreement with other closures. MD starting in conformation I did not transition to II or III, with structures in a single cluster for all flanking basepairs except for G-C where 22% of the structures were defined by a C3'-endo sugar pucker for G4 and G4∗. Bases G4 and G4∗ were also syn around the glycosidic bond for all flanking pairs, whereas G6 and G6∗ transitioned between syn and anti for C-G- and A-U-flanked simulations, consistent with NMR.