The ion atmosphere is a critical structural, dynamic, and energetic component of nucleic acids that profoundly affects their interactions with proteins and ligands. Experimental methods that “count” the number of ions thermodynamically associated with the ion atmosphere allow dissection of energetic properties of the ion atmosphere, and thus provide direct comparison to theoretical results. Previous experiments have focused primarily on the cations that are attracted to nucleic acid polyanions, but have also showed that anions are excluded from the ion atmosphere. Herein, we have systematically explored the properties of anion exclusion, testing the zeroth-order model that anions of different identity are equally excluded due to electrostatic repulsion. Using a series of monovalent salts, we find, surprisingly, that the extent of anion exclusion and cation inclusion significantly depends on salt identity. The differences are prominent at higher concentrations and mirror trends in mean activity coefficients of the electrolyte solutions. Salts with lower activity coefficients exhibit greater accumulation of both cations and anions within the ion atmosphere, strongly suggesting that cation–anion correlation effects are present in the ion atmosphere and need to be accounted for to understand electrostatic interactions of nucleic acids. To test whether the effects of cation–anion correlations extend to nucleic acid kinetics and thermodynamics, we followed the folding of P4–P6, a domain of the Tetrahymena group I ribozyme, via single-molecule fluorescence resonance energy transfer in solutions with different salts. Solutions of identical concentration but lower activity gave slower and less favorable folding. Our results reveal hitherto unknown properties of the ion atmosphere and suggest possible roles of oriented ion pairs or anion-bridged cations in the ion atmosphere for electrolyte solutions of salts with reduced activity. Consideration of these new results leads to a reevaluation of the strengths and limitations of Poisson–Boltzmann theory and highlights the need for next-generation atomic-level models of the ion atmosphere.

Electrostatics are central to all aspects of nucleic acid behavior, including their folding, condensation, and binding to other molecules, and the energetics of these processes are profoundly influenced by the ion atmosphere that surrounds nucleic acids. Given the highly complex and dynamic nature of the ion atmosphere, understanding its properties and effects will require synergy between computational modeling and experiment. Prior computational models and experiments suggest that cation occupancy in the ion atmosphere depends on the size of the cation. However, the computational models have not been independently tested, and the experimentally observed effects were small. Here, we evaluate a computational model of ion size effects by experimentally testing a blind prediction made from that model, and we present additional experimental results that extend our understanding of the ion atmosphere. Giambasu et al. developed and implemented a three-dimensional reference interaction site (3D-RISM) model for monovalent cations surrounding DNA and RNA helices, and this model predicts that Na+ would outcompete Cs+ by 1.8–2.1-fold; i.e., with Cs+ in 2-fold excess of Na+ the ion atmosphere would contain an equal number of each cation (Nucleic Acids Res.2015, 43, 8405). However, our ion counting experiments indicate that there is no significant preference for Na+ over Cs+. There is an ∼25% preferential occupancy of Li+ over larger cations in the ion atmosphere but, counter to general expectations from existing models, no size dependence for the other alkali metal ions. Further, we followed the folding of the P4–P6 RNA and showed that differences in folding with different alkali metal ions observed at high concentration arise from cation–anion interactions and not cation size effects. Overall, our results provide a critical test of a computational prediction, fundamental information about ion atmosphere properties, and parameters that will aid in the development of next-generation nucleic acid computational models.

The composition of the ion atmosphere surrounding nucleic acids affects their folding, condensation and binding to other molecules. It is thus of fundamental importance to gain predictive insight into the formation of the ion atmosphere and thermodynamic consequences when varying ionic conditions. An early step toward this goal is to benchmark computational models against quantitative experimental measurements. Herein, we test the ability of the three dimensional reference interaction site model (3D-RISM) to reproduce preferential interaction parameters determined from ion counting (IC) experiments for mixed alkali chlorides and dsDNA. Calculations agree well with experiment with slight deviations for salt concentrations >200 mM and capture the observed trend where the extent of cation accumulation around the DNA varies inversely with its ionic size. Ion distributions indicate that the smaller, more competitive cations accumulate to a greater extent near the phosphoryl groups, penetrating deeper into the grooves. In accord with experiment, calculated IC profiles do not vary with sequence, although the predicted ion distributions in the grooves are sequence and ion size dependent. Calculations on other nucleic acid conformations predict that the variation in linear charge density has a minor effect on the extent of cation competition.

Highly reproducible and fast potential-assisted immobilization of single-stranded (ss)DNA on gold surfaces is achieved by applying a pulse-type potential modulation. The desired DNA coverage can be obtained in a highly reproducible way within minutes. Understanding the underlying processes occurring during potential-assisted ssDNA immobilization is crucial. We propose a model that considers the role of ions surrounding the DNA strands, the distance dependence of the applied potentials within the electrolyte solution, and most importantly the shift of the potential of zero charge during the immobilization due to the surface modification with DNA. The control of the surface coverage of ssDNA as well as the achieved speed and high reproducibility are seen as prerequisites for improved DNA-based bioassays.

Determination of DNA hybridization at electrode surfaces modified with thiol-tethered single-stranded DNA (ssDNA) capture probes and co-assembled with short-chain thiol derivatives using electrochemical impedance spectroscopy requires a careful design of the electrode/electrolyte interface as well as an in-depth understanding of the processes at the interface during DNA hybridization. The influence of the electrode potential, the ssDNA coverage, the ionic strength, the nature of the thiol derivative and especially the Debye length are shown to have a significant impact on the impedance spectra. A mixed monolayer comprising--in addition to the ssDNA capture probe--both mercaptohexanol (MCH) and mercaptopropionic acid (MPA) is suggested as an interface design which allows a high efficiency of the DNA hybridization concomitantly with a reliable modulation of the charge-transfer resistance of the electrode upon hybridization.

Actinomycin D or proflavine which are known to intercalate within the helix of double-stranded DNA (dsDNA) are used as label-free control to unequivocally prove complementary DNA hybridization by means of electrochemical impedance spectroscopy (EIS). Based on a carefully designed interface comprising a thiol-tethered (20mer) oligonucleotide capture probe which forms a self-assembled monolayer on a gold electrode together with a short chain hydroxyl-terminated alkylthiol, formation of dsDNA can be monitored by an increase of the charge-transfer resistance of a free-diffusing negatively charged redox species ([Fe(CN) 6 ] 3À/4À ). The increase of the charge transfer resistance due to complementary hybridization was about 10 times from the unmodified Au surface to the dsDNA modified electrode. Specific interaction of intercalators with dsDNA leads to a decrease in charge transfer resistance due to the conformational changes in the dsDNA monolayer and partial charge compensation caused by the positively charged intercalators. No shift in the charge transfer resistance was observed in case of incubation of a ssDNA surface with intercalators or when hybridization was invoked using a noncomplementary DNA sequence. Thus, hybridization can be unambiguously detected using EIS by first recording the increase in charge-transfer resistance due to hybridization with the matching target strand followed by recording a decrease in charge transfer resistance caused by intercalation. Nonspecific adsorption can hence be doubtlessly excluded as a reason for the observed changes in the impedance spectrum.

RNAs are one of the most charged polyelectrolytes in nature, and understanding their electrostatics is fundamental to their structure and biological functions. An effective way to characterize the electrostatic field generated by nucleic acids is to quantify interactions between nucleic acids and ions that surround the molecules. These ions form a loosely associated cloud referred to as an ion atmosphere. Although theoretical and computational studies can describe the ion atmosphere around RNAs, benchmarks are needed to guide the development of these approaches, and experiments to date that read out RNA-ion interactions are limited. Here, we present ion counting studies to quantify the number of ions surrounding well-defined model systems of RNA and DNA duplexes. We observe that the RNA duplex attracts more cations and expels fewer anions compared to the DNA duplex, and the RNA duplex interacts significantly stronger with the divalent cation Mg 2þ , despite their identical total charge. These experimental results suggest that the RNA duplex generates a stronger electrostatic field than DNA, as is predicted based on the structural differences between their helices. Theoretical calculations using a nonlinear Poisson-Boltzmann equation give excellent agreement with experiments for monovalent ions but underestimate Mg 2þ-DNA and Mg 2þ-RNA interactions by 20%. These studies provide needed stringent benchmarks to use against other all-atom theoretical models of RNA-ion interactions, interactions that likely must be accurately accounted for in structural, dynamic, and energetic terms to confidently model RNA structure, interactions, and function.

In eukaryotes, a first step towards the nuclear DNA compaction process is the formation of a nucleosome, which is comprised of negatively charged DNA wrapped around a positively charged histone protein octamer. Often, it is assumed that the complexation of the DNA into the nucleosome completely attenuates the DNA charge and hence the electrostatic field generated by the molecule. In contrast, theoretical and computational studies suggest that the nucleosome retains a strong, negative electrostatic field. Despite their fundamental implications for chromatin organization and function, these opposing views of nucleosome electrostatics have not been experimentally tested. Herein, we directly measure nucleosome electrostatics and find that while nucleosome formation reduces the complex charge by half, the nucleosome nevertheless maintains a strong negative electrostatic field. Our studies highlight the importance of considering the polyelectrolyte nature of the nucleosome and its impact on processes ranging from factor binding to DNA compaction.