Why double-stranded RNA resists condensation

Published online 14 August 2014 Nucleic Acids Research, 2014, Vol. 42, No. 16 10823–10831 doi: 10.1093/nar/gku756 Why double-stranded RNA resists condensation Igor S. Tolokh1,† , Suzette A. Pabit2,† , Andrea M. Katz2 , Yujie Chen2 , Aleksander Drozdetski3 , Nathan Baker4 , Lois Pollack2,* and Alexey V. Onufriev1,3,* 1 Department of Computer Science, Virginia Tech, Blacksburg, VA 24061, USA, 2 School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853-3501, USA, 3 Department of Physics, Virginia Tech, Blacksburg, VA 24061, USA and 4 Applied Statistics and Computational Modeling Group, Pacific Northwest National Laboratory, Richland, WA 99352, USA Received May 13, 2014; Revised July 28, 2014; Accepted August 7, 2014 ABSTRACT INTRODUCTION Highly charged DNA molecules are expected to repel each other, yet can be condensed by certain multivalent ions into structured aggregates (1–3). The condensation phenomenon is biologically important. Compaction of anionic DNA and RNA molecules by oppositely charged cationic agents enables efficient packaging of genetic material inside living cells and viruses (4–7). In vitro experiments on DNA * To whom correspondence should be addressed. Tel: +1 540 231 4237; Fax: +1 540 231 6075; Email: Correspondence may also be addressed to Lois Pollack. Tel: +1 607 255 8695; Fax: +1 607 255 7658; Email: . † The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors.  C The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 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. in aqueous solution revealed that the cation-induced condensation requires the ion valence to be +3 or higher (1,8). Trivalent cations, such as cobalt(III) hexammine (CoHex) or spermidine, can effectively condense DNA while divalent inorganic cations (e.g. Mg2+ ) alone cannot, suggesting the significant contribution of electrostatic interactions to the counterintuitive effective attraction between nucleic acids (NA). Over the years, a number of experimental and theoretical studies have been carried out (9–28) with the goal of providing a general physical picture of the condensation phenomenon. A commonly accepted view has emerged: the effective attraction is mainly due to electrostatic contributions. However, the general picture as well as the atomistic mechanism of NA condensation, including the role of hydration forces (12,29) or multivalent counterion correlations (14,26), are still incomplete and cannot be fully explained. Particularly puzzling are our recent experimental results which show that double-stranded (ds) RNA helices resist condensation under conditions where short DNA duplexes condense readily (30). The results are somewhat paradoxical since the multivalent counterions––whose attractive interactions with the oppositely charged duplex are key to the condensation––are expected (31) to bind more strongly to RNA than to DNA. The striking difference between DNA and RNA condensation has sparked renewed efforts to understand the molecular mechanism of NA condensation (32) beyond existing models (13,33–35) and phenomenological theories (27) based on simplified NA geometries. Because dsDNA is typically found in B-type helical forms, while dsRNA stays in A-like forms, it was suggested that counterion distributions around the differing helical forms may explain the difference in condensation behavior of DNA and RNA (30). However, atomically detailed CoHex distributions are hard to measure in solution samples. In principle, all-atom explicit solvent molecular dynamics (MD) simulations (36–43) can provide the necessary details of NA interaction with multivalent ions, including ion dis- 10824 Nucleic Acids Research, 2014, Vol. 42, No. 16 tributions around NA, as well as preferred ion binding sites and their occupancies. To ensure equilibration of multivalent ion distributions, especially around RNA, such simulations will have to go well beyond the time scale of tens of nanoseconds––the longest reported atomistic MD simulation to date of small DNA fragments interacting with trior tetravalent ions in solution (39). To uncover the mechanism of RNA resistance to condensation and to determine key factors responsible for different NA condensation propensities, we studied CoHex-induced condensation and CoHex binding properties of four short NA helical constructs using experiments and experimentally guided atomistic MD simulations on appropriate time scales. MATERIALS AND METHODS Materials CoHex-induced NA condensation as monitored by UV absorption The NA constructs were dialyzed in 20 mM NaCl and 1 mM pH 7.0 Na-MOPS buffer and separated in 40 ␮M 100 ␮l aliquots. Each aliquot was spiked with 5 ␮l of concentrated CoHex and incubated at 4◦ C for 2 h. The aliquots were centrifuged at 10 000 revolutions per minute for 10 min and the supernatant was separated for absorption spectroscopy measurements. The absorption spectra were recorded from 220 to 600 nm using a Cary 50 spectrophotometer. The optical density at 260 nm (OD260 ) was monitored to determine the amount of NA left in the solution. The recorded value was normalized using the OD260 of the sample with no added CoHex and reported in Figure 1 as the fraction of soluble sample in the supernatant. The error bars shown were determined by propagation of errors due to baseline differences at OD600 and presence of residual CoHex at OD473 . Circular dichroism (C (...truncated)