RNA and its ionic cloud: solution scattering experiments and atomically detailed simulations

Abstract

RNA molecules play critical roles in many cellular processes. Traditionally viewed as genetic messengers, RNA molecules were recently discovered to have diverse functions related to gene regulation and expression. RNA also has great potential as a therapeutic and a tool for further investigation of gene regulation. Metal ions are an integral part of RNA structure and should be considered in any experimental or theoretical study of RNA. Here, we report a multidisciplinary approach that combines anomalous small-angle x-ray scattering and molecular-dynamics (MD) simulations with explicit solvent and ions around RNA. From experiment and simulation results, we find excellent agreement in the number and distribution of excess monovalent and divalent ions around a short RNA duplex. Although similar agreement can be obtained from a continuum description of the solvent and mobile ions (by solving the Poisson-Boltzmann equation and accounting for finite ion size), the use of MD is easily extended to flexible RNA systems with thermal fluctuations. Therefore, we also model a short RNA pseudoknot and find good agreement between the MD results and the experimentally derived solution structures. Surprisingly, both deviate from crystal structure predictions. These favorable comparisons of experiment and simulations encourage work on RNA in all-atom dynamic models.

Figure 1

Number of cations in a cylindrical volume of radius r, where r is the distance from the center of the A-form RNA duplex's long axis, is shown for simulations of (a) 0.1 M RbCl (blue, top panel) and (b) 0.1 M SrCl₂ (red, bottom panel). Open points are the total number of cations in a cylindrical volume with radius r, and filled points are the number of cations when the bulk contribution is subtracted from the total number. The horizontal lines are the experimental results for the number of excess ions, with error bars shown at the ends of the lines. We obtained these values using ASAXS to count the number of excess cations around a 25-bp RNA duplex in 0.1 M RbCl (blue dashed) or 0.1 M SrCl₂ (red solid) aqueous solutions (see text for details).

Figure 2

(a) Radial concentration profiles of cations as a function of r, the radial distance from the center of the RNA long axis, are computed from the simulations for four different aqueous solutions. The distance of the phosphates from the central axis is ∼11 Å. Shown here are 0.1 M Na⁺ (blue), 0.1 M Rb⁺ (black), 0.1 M Sr²⁺ (red), and 0.2 M Mg²⁺ (green) distributions. The lines are guides to the eye. Error bars are calculated by grouping the data into three sets and taking the average and standard deviation (see Supporting Material for details). (b) The radial distribution of chloride ions for cations shown in part a are displayed here, using the same color code.

Figure 3

ASAXS signal of 0.1 M (a) Rb⁺ ions (blue points, top panel) and (b) Sr⁺² (red points, bottom panel) around an A-form RNA duplex is plotted as a function of momentum transfer (see text for more details). The spectrum is compared with ASAXS profiles computed from MD simulation data of 0.1 M RbCl (blue solid line, top panel) and 0.1 M SrCl₂ (red solid line, bottom panel). The discrepancy at low q (q < 0.05 Å⁻¹) seen in the lower panel is attributed to structure factor effects from duplex end-to-end stacking that does not manifest in the MD simulations.

Figure 4

Measured scattering profiles of a 28-nt RNA pseudoknot from BWYV (PDB ID 437D) in 0.1 M NaCl solution at room temperature are shown as Kratky plots (open circles). The solid line is the computed spectrum from the MD simulations, sampling many configurations of the RNA and ion distributions in the vicinity of the folded state. A Kratky plot computed from the PDB structure is shown as a dashed line. All plots are normalized to their maximum value. The inset compares the SAXS profiles from the experiment (circles) and MD simulation (line).

Figure 5

Representative configurations from the MD simulations of the BWYV pseudoknot are overlaid on the crystal structure 437D (yellow-lighter). We chose these representative configurations by clustering the simulation data to four clusters via a k-means algorithm. Each configuration shown represents one of the centers of these four clusters. The dashed lines are the distances between the 5′ end of the RNA in the crystal structure and the MD configuration.