High-resolution reversible folding of hyperstable RNA tetraloops using molecular dynamics simulations

Abstract

We report the de novo folding of three hyperstable RNA tetraloops to 1-3 Å rmsd from their experimentally determined structures using molecular dynamics simulations initialized in the unfolded state. RNA tetraloops with loop sequences UUCG, GCAA, or CUUG are hyperstable because of the formation of noncanonical loop-stabilizing interactions, and they are all faithfully reproduced to angstrom-level accuracy in replica exchange molecular dynamics simulations, including explicit solvent and ion molecules. This accuracy is accomplished using unique RNA parameters, in which biases that favor rigid, highly stacked conformations are corrected to accurately capture the inherent flexibility of ssRNA loops, accurate base stacking energetics, and purine syn-anti interconversions. In a departure from traditional quantum chemistrycentric approaches to force field optimization, our parameters are calibrated directly from thermodynamic and kinetic measurements of intra- and internucleotide structural transitions. The ability to recapitulate the signature noncanonical interactions of the three most abundant hyperstable stem loop motifs represents a significant milestone to the accurate prediction of RNA tertiary structure using unbiased all-atom molecular dynamics simulations.

Keywords: RNA folding; molecular simulations.

Fig. 1.

Tetraloop folding simulations. (A) Histograms of GCAA rmsd vs. temperature. The folded fraction is taken as rmsd < 0.4 nm. (B) Number of folded replicas vs. time. UUCG and CUUG both equilibrate within 100 ns, whereas GCAA requires 275 ns before steady state is reached. (C) Fraction of each 0.5-ns block spent in the folded state for all 64 replicas of the GCAA folding simulation. (D) Fraction folded vs. temperature for all three tetraloop sequences.

Fig. 2.

Folded state predictions (colored) vs. experimental structure (gray) for all three hyperstable tetraloops and their rmsd values from the experimentally determined structures. Predictions are centroids from the most populated cluster of the trajectory visiting the lowest REMD temperature. Nucleosides are colored as red (guanine), green (cytosine), yellow (adenine), and cyan (uracil).

Fig. 3.

Tetraloop folding pathways. Two alternate folding pathways are observed for all three hyperstable tetraloops. Thick and thin arrows depict rapid and slow transitions, respectively. Bases are colored the same as in Fig. 2. (A) UUCG folds rapidly if G_L4 is already in syn before collapse, which is required to form the trans-GU wobble base pair (lower pathway). In contrast, misfolds containing the anti-G_L4 must flip G_L4 out of the loop to access the syn conformation and then flip back in to pair with U_L4 (upper pathway). (B) GCAA folds rapidly when G_L1 correctly pairs with A_L4 after loop collapse to form a sheared base pair (lower pathway), but it can also form a nonnative G_L1-A_L3 base pair (upper pathway). A_L3 must flip out before the native G_L1-A_L4 base pair can form. (C) The CUUG tetraloop rapidly folds when the C_L1-G_L4 base pair is preformed before collapse (lower pathway); otherwise, C_L1 is initially flipped out and must flip back into the loop to reach the native state (upper pathway).