Insights into Stability and Folding of GNRA and UNCG Tetraloops Revealed by Microsecond Molecular Dynamics and Well-Tempered Metadynamics

Abstract

RNA hairpins capped by 5'-GNRA-3' or 5'-UNCG-3' tetraloops (TLs) are prominent RNA structural motifs. Despite their small size, a wealth of experimental data, and recent progress in theoretical simulations of their structural dynamics and folding, our understanding of the folding and unfolding processes of these small RNA elements is still limited. Theoretical description of the folding and unfolding processes requires robust sampling, which can be achieved by either an exhaustive time scale in standard molecular dynamics simulations or sophisticated enhanced sampling methods, using temperature acceleration or biasing potentials. Here, we study structural dynamics of 5'-GNRA-3' and 5'-UNCG-3' TLs by 15-μs-long standard simulations and a series of well-tempered metadynamics, attempting to accelerate sampling by bias in a few chosen collective variables (CVs). Both methods provide useful insights. The unfolding and refolding mechanisms of the GNRA TL observed by well-tempered metadynamics agree with the (reverse) folding mechanism suggested by recent replica exchange molecular dynamics simulations. The orientation of the glycosidic bond of the GL4 nucleobase is critical for the UUCG TL folding pathway, and our data strongly support the hypothesis that GL4-anti forms a kinetic trap along the folding pathway. Along with giving useful insight, our study also demonstrates that using only a few CVs apparently does not capture the full folding landscape of the RNA TLs. Despite using several sophisticated selections of the CVs, formation of the loop appears to remain a hidden variable, preventing a full convergence of the metadynamics. Finally, our data suggest that the unfolded state might be overstabilized by the force fields used.

Free Energy Landscape of GAGA and UUCG RNA Tetraloops

“…2 While simulations initialized in the vicinity of the native state are stable on short time-scales under a variety of simulation conditions, [3][4][5][6][7][8] more recent works strongly suggest that these systems are not correctly modeled by the current Amber force field. [9][10][11][12][13][14] Although different improvements have been proposed, 15 there is growing evidence that none of the available corrections are able to capture the crucial non-canonical interactions present in these tetraloops. 12,13 Despite their small size, an ergodic sampling of these systems requires substantial computational resources, in the order of hundreds of µs using massively parallel simulations.…”

Free-energy landscape of a hyperstable RNA tetraloop

“…Additional corrections will be necessary in future modifications to the AMBER force field. Characterizing the free-energy landscapes of RNA tetraloops has long been a goal for RNA molecular simulations and has been the subject of multiple studies on different loop systems using a variety of molecular dynamics techniques (25,26,31,42,49,50). In this study, we have determined that the free-energy landscape of an RNA tetraloop can be rigorously characterized through extensive REMD simulations and that analysis of multiple global and internal degrees of freedom can describe a plethora of configurations associated with these landscapes.…”

Revisiting GNRA and UNCG folds: U-turns versus Z-turns in RNA hairpin loops

“…Efforts to fold these tetranucleotide sequences by molecular dynamics simulations are currently only partially successful, although significant progress has been made in that direction (Kührova et al 2013;Haldar et al 2015;Miner et al 2016). Such modeling attempts have now to face new challenges: finding not only one, but two or more folds, while grasping their relationship with the environment.…”