Divalent counterion-induced condensation of triple-strand DNA

Abstract

Understanding and manipulation of the forces assembling DNA/RNA helices have broad implications for biology, medicine, and physics. One subject of significance is the attractive force between dsDNA mediated by polycations of valence ≥ 3. Despite extensive studies, the physical origin of the "like-charge attraction" remains unsettled among competing theories. Here we show that triple-strand DNA (tsDNA), a more highly charged helix than dsDNA, is precipitated by alkaline-earth divalent cations that are unable to condense dsDNA. We further show that our observation is general by examining several cations (Mg(2+), Ba(2+), and Ca(2+)) and two distinct tsDNA constructs. Cation-condensed tsDNA forms ordered hexagonal arrays that redissolve upon adding monovalent salts. Forces between tsDNA helices, measured by osmotic stress, follow the form of hydration forces observed with condensed dsDNA. Probing a well-defined system of point-like cations and tsDNAs with more evenly spaced helical charges, the counterintuitive observation that the more highly charged tsDNA (vs. dsDNA) is condensed by cations of lower valence provides new insights into theories of polyelectrolytes and the biological and pathological roles of tsDNA. Cations and tsDNAs also hold promise as a model system for future studies of DNA-DNA interactions and electrostatic interactions in general.

Fig. 1.

Small angle x-ray diffraction (SAXD) profiles of the poly(AT*T) and RY*R-21 triplexes. I(Q) is the scattering intensity, with , where 2θ is the scattering angle and λ is the X-ray wavelength. Note that a linear background has been subtracted to show the peaks in B–D. (A) Typical raw image and the integrated profile of condensed poly(AT*T) arrays. Some higher-order peaks can be identified as weak rings, and they can be indexed with a 2D hexagonal lattice. (B) SAXD peak profiles of as-annealed, nuclease S1 digested, and DNase I digested poly(AT*T) tsDNA. (C) SAXD peak profiles of poly(AT*T) triplexes condensed by divalent cations Mg²⁺, Ba²⁺, and Ca²⁺ corresponding to interaxial spacing of 29.8, 30.2, and 29.6 Å, respectively. (D) SAXD profiles of RY*R-21 triplexes condensed by divalent cations Mg²⁺, Ba²⁺, and Ca²⁺ corresponding to interaxial spacing of 29.8, 30.1, and 29.3 Å respectively.

Fig. 2.

Comparison of tsDNA and dsDNA condensed by 2 mM spermine⁴⁺. (A) Poly(AT*T) triplex vs. poly(AT) duplex. The “poly(AT*T)+poly(AT)” is a mixture of the two helices of the same weight ratio. (B) The RY*R-21 triplex versus RY-21 duplex. The “RY*R-21+RY-21” is a mixture of the two helices of the same weight ratio.

Fig. 3.

Temperature dependences of the interaxial spacings of condensed tsDNA and CB-dsDNA. Co(NH₃)₆Cl₃ is the trivalent cobalt³⁺ hexammine chloride. Error bars for DNA interaxial spacings are around 0.1 Å and smaller than symbol sizes.

Fig. 4.

The inter-DNA pressure (Π) vs. spacing curves of the poly(AT*T) triplex and CB-dsDNA. The ↓ indicates the spacing of poly(AT*T) at 20 mM MgCl₂ at 20 °C at zero applied pressure. Note the similarities in the distance dependence and shape in spite of different cations and DNA helices.