Both helix topology and counterion distribution contribute to the more effective charge screening in dsRNA compared with dsDNA

Abstract

The recent discovery of the RNA interference mechanism emphasizes the biological importance of short, isolated, double-stranded (ds) RNA helices and calls for a complete understanding of the biophysical properties of dsRNA. However, most previous studies of the electrostatics of nucleic acid duplexes have focused on DNA. Here, we present a comparative investigation of electrostatic effects in RNA and DNA. Using resonant (anomalous) and non-resonant small-angle X-ray scattering, we characterized the charge screening efficiency and counterion distribution around short (25 bp) dsDNA and RNA molecules of comparable sequence. Consistent with theoretical predictions, we find counterion mediated screening to be more efficient for dsRNA than dsDNA. Furthermore, the topology of the RNA A-form helix alters the spatial distribution of counterions relative to B-form DNA. The experimental results reported here agree well with ion-size-corrected non-linear Poisson-Boltzmann calculations. We propose that differences in electrostatic properties aid in selective recognition of different types of short nucleic acid helices by target binding partners.

Figure 1.

DNA and RNA SAXS profiles I(q) compared with form factors I₀(q). Curves that fall below the form factor indicate repulsive behavior while those above the form factor denote attraction. Nucleic acid solutions are (a) in bulk salt concentration of 3 mM MgCl₂, with matching DNA and RNA concentrations of 0.6 mM; (b) in 6 mM MgCl₂, [DNA] = [RNA] = 1.1 mM; and (c) in 16 mM MgCl₂, [DNA] = [RNA] = 0.6 mM. Note the data in (b) were acquired at higher nucleic acid concentration than that of (a) and (c) to emphasize differences between DNA and RNA. SAXS curves were normalized to each other at 0:08 < q < 0:13 A Å⁻¹ to allow direct comparison of scattering profiles. In this regime, under all conditions probed, the SAXS profiles are identical to the computed form factor, I₀(q), the scattering profile of a non-interacting molecule. The computed form factors match, within error, the experimental form factors measured at very low nucleic acid concentrations, where interparticle interference effects are negligible.

Figure 2.

DNA and RNA second virial coefficients A₂ as a function of [Mg²⁺]. A₂ provides a measure of the strength of intermolecular interactions. Repulsion is lost and the onset of attraction occurs at lower bulk ion concentrations in RNA than in DNA. The attractive regime (A2 < 0) is shaded gray to aid the eye.

Figure 3.

Distibution of ions around DNA and RNA. CRYSOL-predicted scattering profiles (a) in the absence of ions. Experimental ASAXS profiles for nucleic acids in (b) 100 mM Rb⁺, and (c) 100 mM Sr²⁺; [DNA] = [RNA] = 0.2 mM. ASAXS curves were normalized using factors from the extrapolated scattering intensity at q = 0, I(0), weighted by the square of the sample molecular weight. I(0) were determined by Guinier analysis of the low energy (Eoff) SAXS curves (53).

Figure 4.

Comparison of experimental and calculated ASAXS profiles of DNA and RNA shown in 100 mM monovalent ions. Calculated anomalous signals using varying probe ion radius (a) 3; (b) 4; and (c) 5 Å, are shown. All curves were normalized by matching at the lowest q to facilitate comparison of experiment to theory.

Figure 5.

Radial Patterson inversion of the anomalous difference signal calculated using as described by Engelman et al. (56), s = q/2π. Ideally, U(R) reports the lengths of vectors correlating nucleic acids with condensed counterions. (a) U(R) of 100 mM Rb⁺ ASAXS data. In addition, U(R) computed from the APBS simulations for varying probe ion radius of (b) 3; (c) 4; and (d) 5 Å are shown. From this representation, it is clear that an ion radius upper bound of 4 Å is necessary to describe the data.

Figure 6.

NLPB-generated ion density as function of distance from the cylindrical axis of DNA and RNA for 100 mM RbCl, probe ion radius of 4 Å. Ions can come closer to the RNA surface through the deeper major groove. Inset shows a comparison of the major groove sizes, DNA and RNA helices are tilted equally to display the grooves. The PDB structures were generated using NAB (37) and drawn using Chimera (60).