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.

Department of Biochemistry and Microbiology University of Chemistry and Technology Prague Technická 3 166 28 Prague 6 Czech Republic

Department of Physical and Macromolecular Chemistry Faculty of Science Charles University Prague Albertov 6 128 43 Prague 2 Czech Republic

Institute of Biophysics Academy of Sciences of the Czech Republic Královopolská 135 612 65 Brno Czech Republic

Institute of Organic Chemistry and Biochemistry and Gilead Science Research Center Academy of Sciences of the Czech Republic Flemingovo nám 2 166 10 Prague 6 Czech Republic

Regional Centre of Advanced Technologies and Materials Department of Physical Chemistry Faculty of Science Palacký University Olomouc tř 17 Listopadu 12 771 46 Olomouc Czech Republic

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