Does water play a structural role in the folding of small nucleic acids?

Abstract

Nucleic acid structure and dynamics are known to be closely coupled to local environmental conditions and, in particular, to the ionic character of the solvent. Here we consider what role the discrete properties of water and ions play in the collapse and folding of small nucleic acids. We study the folding of an experimentally well-characterized RNA hairpin-loop motif (sequence 5'-GGGC[GCAA]GCCU-3') via ensemble molecular dynamics simulation and, with nearly 500 micros of aggregate simulation time using an explicit representation of the ionic solvent, report successful ensemble folding simulations with a predicted folding time of 8.8(+/-2.0) micros, in agreement with experimental measurements of approximately 10 micros. Comparing our results to previous folding simulations using the GB/SA continuum solvent model shows that accounting for water-mediated interactions is necessary to accurately characterize the free energy surface and stochastic nature of folding. The formation of the secondary structure appears to be more rapid than the fastest ionic degrees of freedom, and counterions do not participate discretely in observed folding events. We find that hydrophobic collapse follows a predominantly expulsive mechanism in which a diffusion-search of early structural compaction is followed by the final formation of native structure that occurs in tandem with solvent evacuation.

FIGURE 1

Schematic and atomic representations of the simulated RNA hairpin with the core region outlined in red.

FIGURE 2

An example of the diverse conformational sampling observed in stem formation is shown. Na⁺ ions near the solute are shown in green (due to the two-dimensional image, actual ion distances from the solute are not well represented). Blue and red arrows indicate native and non-native basepairing before proper alignment. Initial collapse is complete within ∼2 ns in this trajectory, yet non-native basepairing is present after 8 ns. At ∼15 ns the stem is fully formed, including one site of significant electrostatic potential binding a hydrated ion (black arrow) that was also observed in simulations of the relaxed native structure.

FIGURE 3

(a) Log probability distributions are shown for 19 folding and 100 nonfolding collapse events. The apparent barrier for specific collapse trajectories is along the R_g,core degree of freedom, and is crossed early in the collapse event. After collapse to near-native core size, desolvation occurs. In contrast, nonspecific collapse events randomly sample a much greater portion of the conformational space with no apparent bulk trend. (b) Hydrophobic collapse in a single trajectory is shown with the dashed vertical line indicating the midpoint of the desolvation transition. Structures preceding, concurrent with, and after this midpoint are shown above the frame for visual clarification. R_g,core reaches its native value at the desolvation midpoint (∼2.2 ns), but significant exposed base surface area remains to be buried, resulting in an expulsionlike mechanism.

FIGURE 4

P_fold versus P_fold plots comparing the implicit and explicit solvent and ion models are shown in frames a–d. Each frame combines the P_fold values for both folding pathways previously detected using the GB/SA continuum solvent. Comparisons between TIP3P and TIP4P explicit water models using sodium and magnesium counterions are shown in e and f.

FIGURE 5

Free energy profiles of the two GB/SA folding pathways as sampled in TIP explicit solvent. The compaction pathway in a is two-state, as predicted using the implicit solvent model. In contrast, the zipping pathway in b appears to include diffusive, downhill folding in explicit solvent, whereas GB/SA sampling predicted a two-state landscape. In both cases, the R_g of non-native conformations is predominantly nativelike.