DNA condensation in two dimensions

Abstract

We have found that divalent electrolyte counterions common in biological cells (Ca(2+), Mg(2+), and Mn(2+) ) can condense anionic DNA molecules confined to two-dimensional cationic surfaces. DNA-condensing agents in vivo include cationic histones and polyamines spermidine and spermine with sufficiently high valence (Z) 3 or larger. In vitro studies show that electrostatic forces between DNA chains in bulk aqueous solution containing divalent counterions remain purely repulsive, and DNA condensation requires counterion valence Z >/= 3. In striking contrast to bulk behavior, synchrotron x-ray diffraction and optical absorption experiments show that above a critical divalent counterion concentration the electrostatic forces between DNA chains adsorbed on surfaces of cationic membranes reverse from repulsive to attractive and lead to a chain collapse transition into a condensed phase of DNA tethered by divalent counterions. This demonstrates the importance of spatial dimensionality to intermolecular interactions where nonspecific counterion-induced electrostatic attractions between the like-charged polyelectrolytes overwhelm the electrostatic repulsions on a surface for Z = 2. This new phase, with a one-dimensional counterion liquid trapped between DNA chains at a density of 0.63 counterions per DNA bp, represents the most compact state of DNA on a surface in vitro and suggests applications in high-density storage of genetic information and organo-metallic materials processing.

Figure 1

Schematic illustration of the force reversal between DNA chains adsorbed on cationic membrane surfaces within the lamellar L_α^c phase. For divalent counterion concentrations M < M* the electrostatic forces (F_e) are repulsive. For M > M* the forces become attractive, which leads to the DNA condensation transition on a surface. In contrast, the electrostatic forces between DNA chains in bulk aqueous solution with divalent counterions are purely repulsive. During the transition, the spacing between the DNA double helices rapidly decreases to a separation of order the diameter of the condensing ions (shown as red spheres). In the condensed state (M > M*) there are ≈0.63 ions/base pair along the DNA.

Figure 2

(a) Synchrotron XRD measurements of the powder isoelectric [ρ = (weight lipid)/(weight DNA) = 2.2, Φ_DOPC = (weight DOPC)/(weight lipid) = mole fraction of DOPC = 0.6] CL-DNA complex samples in the presence of MgCl₂. d_DNA abruptly changes from 47 Å (set by Φ_DOPC) at low MgCl₂ concentrations (M) to 28.9 Å above M = M* = 48.2 mM. Also, the complex periodicity d increases to 70 ± 1 Å for M > M*. (b) Similar XRD measurements in the presence of CoCl₂ show that Co²⁺ ions also cause a condensation transition of the 2D DNA arrays but at smaller M* ≈ 24 mM. The (003) peak appears for M > M* in a and b, while the (004) is visible below and above M*. This is because the (003) peak in the lipid-DNA lamellar structure factor is near a zero crossing of the form factor. For M > M*, the screening of the head groups in the presence of the trapped counterions leads to a decrease in the area per charged head group. To match the change in the area per head, the area per tail decreases through chain stretching, which leads to a different position for the zero-crossing of the form factor. (c) Variation of the d_DNA with the concentrations of four different divalent salts.

Figure 3

The number of bound Co²⁺ counterions per DNA base pair as a function of the divalent salt CoCl₂ concentration. The counterion concentration within the isoelectric Φ_DOPC = 0.6 CL-DNA complexes with CoCl₂ was quantified directly by measuring the supernatant optical density at 512 nm.

Figure 4

(a) Synchrotron XRD measurements of the complex supernatant below (●) and above (○) the condensation transition in the presence of MnCl₂. For M > M* the XRD pattern shows the existence of a multilamellar lipid phase of highly charged lipids expelled from the complexes during the 2D DNA condensation. (b) Wide-angle x-ray measurements of the complexes with CoCl₂. Solid arrow indicates the peak caused by the fluid lipid chain packing and dashed arrow indicates the peak of water. (c) The onset of condensation occurs at lower values of MgCl₂ concentration M* as the initial DNA interaxial spacing d_DNA is decreased from 50.3 Å to 47.0 Å to 39.5 Å for Φ_DOPC = 0.7, 0.6, and 0.5, respectively.

Figure 5

(a) Synchrotron XRD measurements of the isoelectric Φ_DOPC = 0.6 complexes with trivalent polyamine spermidine chloride. Spermidine causes DNA condensation in 2D (DNA correlation peak indicated with dashed lines) at 11.9 mM, and at higher concentration also removes DNA out of the complexes into a coexisting phase of spermidine-condensed 3D DNA with hexagonal symmetry (solid arrow at 14 mM). Solid line shows a SAXS scan of pure bulk DNA condensed with ≈14 mM of spermidine (without lipids) and proves that the extra peak in 14 mM complex scan is caused by the coexisting spermidine-DNA phase. (b) Variation of d_DNA in complexes with spermidine and spermine. Also shown is the DNA spacing a in the bulk spermidine and spermine condensed DNA phase.