Probing counterion modulated repulsion and attraction between nucleic acid duplexes in solution

Abstract

Understanding biological and physical processes involving nucleic acids, such as the binding of proteins to DNA and RNA, DNA condensation, and RNA folding, requires an understanding of the ion atmosphere that surrounds nucleic acids. We have used a simple model DNA system to determine how the ion atmosphere modulates interactions between duplexes in the absence of specific metal ion-binding sites and other complicated interactions. In particular, we have tested whether the Coulomb repulsion between nucleic acids can be reversed by counterions to give a net attraction, as has been proposed recently for the rapid collapse observed early in RNA folding. The conformation of two DNA duplexes tethered by a flexible neutral linker was determined in the presence of a series of cations by small angle x-ray scattering. The small angle x-ray scattering profiles of two control molecules with distinct shapes (a continuous duplex and a mimic of the compact DNA) were in good agreement with predictions, establishing the applicability of this approach. Under low-salt conditions (20 mM Na+), the tethered duplexes are extended because of a Coulombic repulsion estimated to be 2-5 kT/bp. Addition of high concentrations of Na+ (1.2 M), Mg2+ (0.6 M), and spermidine3+ (75 mM) resulted in electrostatic relaxation to a random state. These results indicate that a counterion-induced attractive force between nucleic acid duplexes is not significant under physiological conditions. An upper limit on the magnitude of the attractive potential under all tested ionic conditions is estimated.

Fig. 1.

The schemes of the model DNA constructs applied herein. (A) The 24-bp duplex, 12_/PEG9/12 and 12_/C3/12.(B) The circular DNA.

Fig. 2.

The three conformational states of the tethered DNA (12_/PEG9/12, Fig. 1 A) distinguished herein.

Fig. 3.

Monitoring conformational states of the tethered DNA duplexes by SAXS. (A) Predicted SAXS profiles of the extended (dotted line), random (solid line), and collapsed (dashed line) states of 12-bp tethered DNA (12_/PEG9/12, Fig. 1A). Scattering intensity I(s) has been multiplied by the scattering angle s to help illustrate SAXS profile differences; Inset shows the unweighted profile with intensity normalized to unity at s = 0. (B) Experimental SAXS profiles of 24-bp duplex DNA (diamonds) and circular DNA (circles) compared to the predicted SAXS profiles of the duplex (dotted line) and circular (solid line) DNA. (Inset) Plots of I(s), normalized as in A. SAXS data were obtained with 0.2 mM DNA in 1.2 M NaCl/100 mM Na-Mops, pH 7.0.

Fig. 4.

Experimental SAXS profiles of the tethered DNA constructs: 12_/PEG9/12 (A); 12_/C3/12 (B); and 80_/PEG9/80 (C) (see Figs. 1 A and 7) in different salt conditions: 20 mM Na⁺ (diamonds), 1.2 M Na⁺ (squares), 0.6 M Mg²⁺ (circles), and 75 mM spermidine³⁺ (×), compared to the predicted SAXS profiles for the extended (dotted line), random (solid line), and collapsed (dashed line) states for each of the tethered duplexes. SAXS profiles were obtained with DNA concentrations of 0.2 mM (12_/PEG9/12), 0.2 mM (12_/C3/12), and 0.015 mM (80_/PEG9/80), in 100 mM Na-Mops, pH 7.0. (Insets) Residuals of experimental SAXS profiles of tethered DNA compared to their corresponding random state predictions, plotted against s from 0 to 0.04 (1/Å).

Fig. 5.

Estimate of repulsive potential between the tethered DNA duplexes in low salt (A) and the maximal repulsion and attraction in high salt (B). Eqs. 1 and 2 were used to determined the values of λ and ΔG_P-P (Insets) or ΔG_DNA-DNA that are consistent with the observed SAXS profiles for the 12_/PEG9/12 in 20 mM Na⁺ (A) or 1.2 M Na⁺ (solid line), 0.6 M Mg²⁺ (dashed line), and 75 mM spermidine³⁺ (dotted line) (B). The shaded areas represent 95% confidence regions for parameters that are consistent with the observed SAXS profiles. The solid arrows indicate the range (25–55 kT) of interhelical repulsion at low salt (A) and the upper limit (≈1.4 kT) in high salt (B), at decay length of 20 Å and 3 Å, respectively, the Debye screening lengths under the low and high salt conditions, respectively (see Supporting Text). The dotted arrow indicates the strongest attractive potential (–1 kT) in multivalent ions for which the decay length is not less than the smallest diameter of one cation (1.3 Å).

Fig. 6.

Estimate of maximal attractive potential between the tethered DNA 12_/PEG9/12 in high salt by the orientation constrained model. The values of ΔG_attr are consistent with data (values give 95% confidence in χ² test) in 0.6 M Mg²⁺ (A) and 75 mM spermidine³⁺ (B), given different cutoffs for dihedral angle θ, and the center-mass distance R_cm [25 Å (circles), 30 Å (squares), and 35 Å (triangles)] between the two DNA helices.