Why double-stranded RNA resists condensation

Abstract

The addition of small amounts of multivalent cations to solutions containing double-stranded DNA leads to inter-DNA attraction and eventual condensation. Surprisingly, the condensation is suppressed in double-stranded RNA, which carries the same negative charge as DNA, but assumes a different double helical form. Here, we combine experiment and atomistic simulations to propose a mechanism that explains the variations in condensation of short (25 base-pairs) nucleic acid (NA) duplexes, from B-like form of homopolymeric DNA, to mixed sequence DNA, to DNA:RNA hybrid, to A-like RNA. Circular dichroism measurements suggest that duplex helical geometry is not the fundamental property that ultimately determines the observed differences in condensation. Instead, these differences are governed by the spatial variation of cobalt hexammine (CoHex) binding to NA. There are two major NA-CoHex binding modes—internal and external—distinguished by the proximity of bound CoHex to the helical axis. We find a significant difference, up to 5-fold, in the fraction of ions bound to the external surfaces of the different NA constructs studied. NA condensation propensity is determined by the fraction of CoHex ions in the external binding mode.

Spermine Condenses DNA, but Not RNA Duplexes

“…In both systems, the spermine nitrogen atoms bind preferentially to the phosphate groups centered 10 Å from the helical axis of the duplex. However, the spermine binding modes vary between the DNA and RNA, likely due to structural differences such as the width of the major groove and the relative orientation of the phosphate group oxygens, and to the more negative electrostatic potential in the major groove of A-form RNA compared with B-form DNA (23,36). The electrostatic potential of each nucleic acid is shown in Fig.…”

“…The spermine/phosphate ratio used here was lower than that used for the mixed-sequence molecules due to condensation of the poly(rA):poly(rU) RNA at the higher ratio. The high NaCl concentration of these samples relative to those used in UV absorption measurements was chosen to prevent condensation of the CD samples, as in (23). Samples were loaded into a 0.01 mm demountable cuvette (0.1 mm path length in the case of poly(rA):poly(rU)).…”

“…After these stages, 600 ns production trajectories were generated for each system using an NVT ensemble and a 2 fs time step. The DNA atoms were restrained with a reduced force constant of 50 kcal/mol/Å 2 in the production stage, whereas RNA atoms were unrestrained, as discussed previously (23). Spermine charge distributions were calculated as the average numbers of spermine nitrogen atoms (where the centers of the spermines' positive charges are located) in 0.25-Å -thick, consecutive cylindrical layers around the duplexes.…”

“…Previously, we studied the condensation of duplex DNA and RNA by the trivalent cation cobalt(III) hexammine (CoHex) and arrived at the surprising conclusion that RNA resists condensation under the same conditions that cause DNA of an equivalent sequence to condense readily (22,23). The RNA duplexes used in that work have a well-defined composition and sequence (see Materials and Methods), containing a mixture of both GC and AU basepairs to imitate physiological RNA.…”

“…This sequence favors duplex formation over stem loops in the individual strands and has a high melting temperature (61 C as calculated with OligoCalc (24)). We compared the experimental results with all-atom molecular-dynamics (MD) simulations to develop a model in which the distribution of bound CoHex ions relates to the condensation propensity: nucleic acids with more CoHex bound in the external binding shell (12)(13)(14)(15)(16) Å from the helical axis) condense most readily, whereas those with more ions bound to the internal shell (7-12 Å from the helical axis) resist condensation (23,25). Although these studies produced interesting experimental findings, they were not biological.…”