Additive Modulation of DNA-DNA Interactions by Interstitial Ions

Quantitative understanding of biomolecular electrostatics, particularly involving multivalent ions and highly charged surfaces, remains lacking. Ion-modulated interactions between nucleic acids provide a model system in which electrostatics plays a dominant role. Using ordered DNA arrays neutralized by spherical cobalt³⁺ hexammine and Mg²⁺ ions, we investigate how the interstitial ions modulate DNA-DNA interactions. Using methods of ion counting, osmotic stress, and x-ray diffraction, we systematically determine thermodynamic quantities, including ion chemical potentials, ion partition, DNA osmotic pressure and force, and DNA-DNA spacing. Analyses of the multidimensional data provide quantitative insights into their interdependencies. The key finding of this study is that DNA-DNA forces are observed to linearly depend on the partition of interstitial ions, suggesting the dominant role of ion-DNA coupling. Further implications are discussed in light of physical theories of electrostatic interactions and like-charge attraction.     

Protein–DNA and ion–DNA interactions revealed through contrast variation SAXS

Understanding how DNA carries out its biological roles requires knowledge of its interactions with biological partners. Since DNA is a polyanionic polymer, electrostatic interactions contribute significantly. These interactions are mediated by positively charged protein residues or charge compensating cations. Direct detection of these partners and/or their effect on DNA conformation poses challenges, especially for monitoring conformational dynamics in real time. Small-angle x-ray scattering (SAXS) is uniquely sensitive to both the conformation and local environment (i.e. protein partner and associated ions) of the DNA. The primary challenge of studying multi-component systems with SAXS lies in resolving how each component contributes to the measured scattering. Here, we review two contrast variation (CV) strategies that enable targeted studies of the structures of DNA or its associated partners. First, solution contrast variation enables measurement of DNA conformation within a protein–DNA complex by masking out the protein contribution to the scattering profile. We review a specific example, in which the real-time unwrapping of DNA from a nucleosome core particle is measured during salt-induced disassembly. The second method, heavy atom isomorphous replacement, reports the spatial distribution of the cation cloud around duplex DNA by exploiting changes in the scattering strength of cations with varying atomic numbers. We demonstrate the application of this approach to provide the spatial distribution of monovalent cations (Na⁺, K⁺, Rb⁺, Cs⁺) around a standard 25-base pair DNA. The CV strategies presented here are valuable tools for understanding DNA interactions with its biological partners.     

Diffusion NMR-based comparison of electrostatic influences of DNA on various monovalent cations

Counterions are important constituents for the structure and function of nucleic acids. Using ⁷Li and ¹³³Cs nuclear magnetic resonance (NMR) spectroscopy, we investigated how ionic radii affect the behavior of counterions around DNA through diffusion measurements of Li⁺ and Cs⁺ ions around a 15-bp DNA duplex. Together with our previous data on ²³Na⁺ and ¹⁵NH₄⁺ ions around the same DNA under the same conditions, we were able to compare the dynamics of four different monovalent ions around DNA. From the apparent diffusion coefficients at varied concentrations of DNA, we determined the diffusion coefficients of these cations inside and outside the ion atmosphere around DNA (D_b and D_f, respectively). We also analyzed ionic competition with K⁺ ions for the ion atmosphere and assessed the relative affinities of these cations for DNA. Interestingly, all cations (i.e., Li⁺, Na⁺, NH₄⁺, and Cs⁺) analyzed by diffusion NMR spectroscopy exhibited nearly identical D_b/D_f ratios despite the differences in their ionic radii, relative affinities, and diffusion coefficients. These results, along with the theoretical relationship between diffusion and entropy, suggest that the entropy change due to the release of counterions from the ion atmosphere around DNA is also similar regardless of the monovalent ion types. These findings and the experimental diffusion data on the monovalent ions are useful for examination of computational models for electrostatic interactions or ion solvation.     

88 Comparison of monovalent and divalent ion distributions around a DNA duplex with molecular dynamic simulation and Poisson–Boltzmann approach

Ion interactions with nucleic acids (both DNA and RNA) are an important and evolving field of investigation. Positively charged cations may interact with highly negatively charged nucleic acids via simple electrostatic interactions to help screen the electrostatic repulsion along the nucleic acids and assist their folding and/or compaction. Cations may also bind at specific sites and become integral parts of the structures, possibly playing important enzymatic roles. Two popular methods for computationally exploring a nucleic acid’s ion atmosphere are atomistic molecular dynamics (MD) simulations and the Poisson–Boltzmann (PB) equation. In general, monovalent ion results obtained from MD simulations and the PB equation agree well with experiment. However, Bai et al. (2007) observed discrepancies between experiment and the PB equation while examining the competitive binding of monovalent and divalent ions, with more significant discrepancies for divalent ions. The goal of this project was to thoroughly investigate monovalent (Na⁺) and divalent (Mg²⁺) ion distributions formed around a DNA duplex with MD simulations and the PB equation. We simulated three different cation concentrations, and matched the equilibrated bulk ion concentration for our theoretical calculations with the PB equation. Based on previous work, our Mg²⁺ ions were fully solvated, the expected state of Mg²⁺ ions when interacting with a duplex, when the production simulations began and remained throughout the simulations (Kirmizialtin, 2010; Robbins, 2012). Na⁺ ion distributions and number of Na⁺ ions within 10?Å of the DNA obtained from our two methods agreed well. However, results differed for Mg²⁺ ions, with a lower number of ions within the cut-off distance obtained from the PB equation when compared to MD simulations. The Mg²⁺ ion distributions around the DNA obtained via the two methods also differed. Based on our results, we conclude that the PB equation will systematically underestimate Mg²⁺ ions bound to DNA, and much of this deviation is attributed to dielectric saturation associated with high valency ions.     

Comparison of monovalent and divalent ion distributions around a DNA duplex with molecular dynamics simulation and a Poisson‐Boltzmann approach

The ion atmosphere created by monovalent (Na⁺) or divalent (Mg²⁺) cations surrounding a B‐form DNA duplex were examined using atomistic molecular dynamics (MD) simulations and the nonlinear Poisson‐Boltzmann (PB) equation. The ion distributions predicted by the two methods were compared using plots of radial and two‐dimensional cation concentrations and by calculating the total number of cations and net solution charge surrounding the DNA. Na⁺ ion distributions near the DNA were more diffuse in PB calculations than in corresponding MD simulations, with PB calculations predicting lower concentrations near DNA groove sites and phosphate groups and a higher concentration in the region between these locations. Other than this difference, the Na⁺ distributions generated by the two methods largely agreed, as both predicted similar locations of high Na⁺ concentration and nearly identical values of the number of cations and the net solution charge at all distances from the DNA. In contrast, there was greater disagreement between the two methods for Mg²⁺ cation concentration profiles, as both the locations and magnitudes of peaks in Mg²⁺ concentration were different. Despite experimental and simulation observations that Mg²⁺ typically maintains its first solvation shell when interacting with nucleic acids, modeling Mg²⁺ as an unsolvated ion during PB calculations improved the agreement of the Mg²⁺ ion atmosphere predicted by the two methods and allowed for values of the number of bound ions and net solution charge surrounding the DNA from PB calculations that approached the values observed in MD simulations. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 834–848, 2014.     

Vesicle Adhesion and Fusion Studied by Small-Angle X-Ray Scattering

We have studied the adhesion state (also denoted by docking state) of lipid vesicles as induced by the divalent ions Ca²⁺ or Mg²⁺ at well-controlled ion concentration, lipid composition, and charge density. The bilayer structure and the interbilayer distance in the docking state were analyzed by small-angle x-ray scattering. A strong adhesion state was observed for DOPC:DOPS vesicles, indicating like-charge attraction resulting from ion correlations. The observed interbilayer separations of ～1.6 nm agree quantitatively with the predictions of electrostatics in the strong coupling regime. Although this phenomenon was observed when mixing anionic and zwitterionic (or neutral) lipids, pure anionic membranes (DOPS) with highest charge density σ resulted in a direct phase transition to a multilamellar state, which must be accompanied by rupture and fusion of vesicles. To extend the structural assay toward protein-controlled docking and fusion, we have characterized reconstituted N-ethylmaleimide-sensitive factor attachment protein receptors in controlled proteoliposome suspensions by small-angle x-ray scattering.     

Predicting Ion Binding Properties for RNA Tertiary Structures

Recent experiments pointed to the potential importance of ion correlation for multivalent ions such as Mg²⁺ ions in RNA folding. In this study, we develop an all-atom model to predict the ion electrostatics in RNA folding. The model can treat ion correlation effects explicitly by considering an ensemble of discrete ion distributions. In contrast to the previous coarse-grained models that can treat ion correlation, this new model is based on all-atom nucleic acid structures. Thus, unlike the previous coarse-grained models, this new model allows us to treat complex tertiary structures such as HIV-1 DIS type RNA kissing complexes. Theory-experiment comparisons for a variety of tertiary structures indicate that the model gives improved predictions over the Poisson-Boltzmann theory, which underestimates the Mg²⁺ binding in the competition with Na⁺. Further systematic theory-experiment comparisons for a series of tertiary structures lead to a set of analytical formulas for Mg²⁺/Na⁺ ion-binding to various RNA and DNA structures over a wide range of Mg²⁺ and Na⁺ concentrations.     

Stabilities in nitromethane of alkali metal ion complexes with dibenzo-18-crown-6 and dibenzo-24-crown-8 and their transfer from nitromethane to other polar solvents

Stability constants for the 1:1 complexes of Na⁺, K⁺, Rb⁺, and Cs⁺ with dibenzo-18-crown-6 (DB18C6) and dibenzo-24-crown-8 (DB24C8) have been determined by conductometry at 25 °C in a poorly solvating solvent, nitromethane. For both the crown ethers, the stability constant decreases with increasing metal ion size, Na⁺ > K⁺ > Rb⁺ > Cs⁺, regardless of the size compatibility between the metal ions and the ligand cavities. A comparison of the results with those in several other solvents (S: acetonitrile, propylene carbonate, water, methanol, and N,N-dimethylformamide) leads to the conclusion that the selectivity sequence of these crown ethers in nitromethane agrees with the intrinsic one in the absence of a solvent. Transfer activity coefficients of the crown ethers and their complexes from nitromethane to S have been determined to evaluate the solute-solvent interactions. It is shown that DB24C8 shields the alkali metal ions more effectively from the solvents than DB18C6 because of the larger number of oxygen atoms and the more flexible structure of DB24C8. Regarding the complexation in nitromethane as a reference, the complex stability and selectivity in S are discussed. The selectivities of these crown ethers in water, methanol, and N,N-dimethylformamide, which apparently obey the size-fit concept, are largely due to the solvation of the free alkali metal ions.     

Studies on Ion Accumulation in Muscle Cells

A comparison is made between the quantitative predictions of equilibrium ionic distribution in living cells according to the membrane theory (Donnan equilibrium) and according to the association-induction hypothesis. This comparison shows that both theories predict competitive effects of one permeant ion on the equilibrium concentration of another permeant ion; but within the limit of experimental accuracy only the association-induction model predicts quantitatively significant specific competition of one specified ion with the accumulation of another specified ion. The equilibrium distributions of K⁺, Rb⁺, and Cs⁺ ions in frog sartorius muscle were studied and quantitatively significant specific competition was demonstrated; these results favor the association-induction hypothesis (adsorption on cell proteins and protein complexes and partial exclusion from cell water). Based on this model we estimated that at 257deg;C, the apparent association constants for K⁺, Rb⁺, and Cs⁺ ion are 665, 756, and 488 (mole/liter)^-1. We found that the total concentration of adsorption sites (no less than 240 mmole/kg of fresh cells) agrees with the analytically determined concentrations of β- and γ-carboxyl groups of muscle cell proteins (260 to 288 mmole/kg).     

Thermodynamics of site-specific small molecular ion interactions with DNA duplex: a molecular dynamics study

The stability and dynamics of a double-stranded DNA (dsDNA) is affected by the preferential occupancy of small monovalent molecular ions. Small metal and molecular ions such as sodium and alkyl ammonium have crucial biological functions in human body, affect the thermodynamic stability of the duplex DNA and exhibit preferential binding. Here, using atomistic molecular dynamics simulations, we investigate the preferential binding of metal ion such as Na⁺ and molecular ions such as tetramethyl ammonium (TMA⁺) and 2-hydroxy-N,N,N-trimethylethanaminium (CHO⁺) to double-stranded DNA. The thermodynamic driving force for a particular molecular ion-DNA interaction is determined by decomposing the free energy of binding into its entropic and enthalpic contributions. Our simulations show that each of these molecular ions preferentially binds to the minor groove of the DNA and the extent of binding is highest for CHO⁺. The ion binding processes are found to be entropically favourable. In addition, the contribution of hydrophobic effects towards the entropic stabilisation (in case of TMA⁺) and the effect of hydrogen bonding contributing to enthalpic stabilisation (in case of CHO⁺) have also been investigated.     

Silver nanoclusters stabilized with denatured fish sperm DNA and the application on trace mercury ions detection

In this study, fluorescent silver nanoclusters (Ag NCs) were synthesized using denatured fish sperm DNA as the template. In contrast to other methods, this method did not use artificial DNA as the template. After their reaction with denatured fish sperm DNA, Ag⁺ ions were reduced by NaBH₄ to form Ag NCs. The Ag NCs showed a strong fluorescence emission at 650 nm when excited at 585 nm. The fluorescence intensity increased fourfold at pH 3.78, controlled with Britton–Robinson buffer solution. The fluorescence of the Ag NCs was quenched in the presence of trace mercury ions (Hg²⁺) in a weakly acidic medium and nitrogen atmosphere. The extent of the fluorescence quenching of Ag NCs strongly depends on the Hg²⁺ ion concentration over a linear range from 2.0 nmol L^?1 to 3.0 μmol L^?1. The detection limit (3σ/k) for Hg²⁺ was 0.7 nmol L^?1. Thus, a sensitive and rapid method was developed for the detection of Hg²⁺ ions.     

Examining the contributions of lipid shape and headgroup charge on bilayer behavior

To better understand bilayer property dependency on lipid electrostatics and headgroup size, we use atomistic molecular dynamics simulations to study negatively charged and neutral lipid membranes. We compare the negatively charged phosphatidic acid (PA), which at physiological pH and salt concentration has a negative spontaneous curvature, with the negatively charged phosphatidylglycerol (PG) and neutrally charged phosphatidylcholine (PC), both of which have zero spontaneous curvature. The PA lipids are simulated using two different sets of partial charges for the headgroup and the varied charge distribution between the two PA systems results in significantly different locations for the Na⁺ ions relative to the water/membrane interface. For one PA system, the Na⁺ ions are localized around the phosphate group. In the second PA system, the Na⁺ ions are located near the ester carbonyl atoms, which coincides with the preferred location site for the PG Na⁺ ions. We find that the Na⁺ ion location has a larger effect on bilayer fluidity properties than lipid headgroup size, where the A_lipid and acyl chain order parameter values are more similar between the PA and PG bilayers that have Na⁺ ions located near the ester groups than between the two PA bilayers.     

Nuclear Magnetic Resonance Study of Mammalian Cell Water: Influence of Water Content and Ionic Environment

The water proton spin-lattice relaxation time (T₁) in mammalian cells and tissues has been measured as a function of external ion concentration and total cell water content. The results can be interpreted in terms of changes in the fractions of bound and unbound water, and changes in the coordination shells of macromolecules due to alterations in macromolecular configuration caused by changes in salt molarity and the amount of water. It is shown that the direct effect of the ions (Na⁺, K⁺, Li⁺, Cl^-) on structuring cellular water, i.e., into ion coordination shells, is small; the main effect of these ions on cellular water structure is an indirect one, resulting from their capability of changing macromolecular coordination shells.     

Contact pairs of RNA with magnesium ions-electrostatics beyond the Poisson-Boltzmann equation

The electrostatic interaction of RNA with its aqueous environment is most relevant for defining macromolecular structure and biological function. The attractive interaction of phosphate groups in the RNA backbone with ions in the water environment leads to the accumulation of positively charged ions in the first few hydration layers around RNA. Electrostatics of this ion atmosphere and the resulting ion concentration profiles have been described by solutions of the nonlinear Poisson-Boltzmann equation and atomistic molecular dynamics (MD) simulations. Much less is known on contact pairs of RNA phosphate groups with ions at the RNA surface, regarding their abundance, molecular geometry, and role in defining RNA structure. Here, we present a combined theoretical and experimental study of interactions of a short RNA duplex with magnesium (Mg²⁺) ions. MD simulations covering a microsecond time range give detailed hydration geometries as well as electrostatics and spatial arrangements of phosphate-Mg²⁺ pairs, including both pairs in direct contact and separated by a single water layer. The theoretical predictions are benchmarked by linear infrared absorption and nonlinear two-dimensional infrared spectra of the asymmetric phosphate stretch vibration which probes both local interaction geometries and electric fields. Contact pairs of phosphate groups and Mg²⁺ ions are identified via their impact on the vibrational frequency position and line shape. A quantitative analysis of infrared spectra for a range of Mg²⁺-excess concentrations and comparison with fluorescence titration measurements shows that on average 20–30% of the Mg²⁺ ions interacting with the RNA duplex form contact pairs. The experimental and MD results are in good agreement. In contrast, calculations based on the nonlinear Poisson-Boltzmann equation fail in describing the ion arrangement, molecular electrostatic potential, and local electric field strengths correctly. Our results underline the importance of local electric field mapping and molecular-level simulations to correctly account for the electrostatics at the RNA-water interface.     

Distinct differences in metal ion specificity of RNA and DNA G-quadruplexes

RNA G-quadruplexes, as their well-studied DNA analogs, require the presence of cations to fold and remain stable. This is the first comprehensive study on the interaction of RNA quadruplexes with metal ions. We investigated the formation and stability of two highly conserved and biologically relevant RNA quadruplex-forming sequences (24nt-TERRA and 18nt-NRAS) in the presence of several monovalent and divalent metal ions, namely Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, NH₄ ⁺, Mg²⁺, Ca²⁺, Sr²⁺, and Ba²⁺. Circular dichroism was used to probe the influence of these metal ions on the folded fraction of the parallel G-quadruplexes, and UV thermal melting experiments allowed to assess the relative stability of the structures in each cationic condition. Our results show that the RNA quadruplexes are more stable than their DNA counterparts under the same buffer conditions. We have observed that the addition of mainly Na⁺, K⁺, Rb⁺, NH₄ ⁺, as well as Sr²⁺ and Ba²⁺ in water, shifts the equilibrium to the folded quadruplex form, whereby the NRAS sequence responds stronger than TERRA. However, only K⁺ and Sr²⁺ lead to a significant increase in the stability of the folded structures, which is consistent with their coordination to the O6 atoms from the G-quartet guanosines. Compared to the respective DNA motives, dNRAS and htelo, the RNA sequences are not stabilized by Na⁺ ions. Finally, the difference in response between NRAS and TERRA, as well as to the corresponding DNA sequences with respect to different metal ions, could potentially be exploited for selective targeting purposes.     

Analysis of salts transport affected by root absorption capacity in surface — irrigated fields in the upper Yellow River basin

In order to analyze the salt transport affected by roots and its effects on soil salinity in an experimental irrigated field newly established in an alluvial valley of the Yellow River in China, spatial distribution of ions contained in waters, soils and crops relevant to these phenomena were evaluated there. During the intensive surveys conducted in year 2007–2008, the Yellow River water, irrigation canal water, groundwater, field soils and crops, etc. were sampled and their chemical characteristics such as electrical conductivity, concentrations of ions Na⁺, Ca²⁺, Mg²⁺, K⁺, Cl⁻, SO₄²⁻and NO₃⁻ were measured. Irrigation seemed to cause increases in the concentrations of ions Na⁺, Cl⁻ and SO₄²⁻ in the groundwater. Although those were also major ions contained in the field soil, the soil was classed as saline but not sodic according to the standard classification. On the other hand, K⁺, which is one of the major essential nutrients for plant growth, was highly concentrated in the crops, while Na⁺ was not concentrated because of crop’s poor ability to absorb it. The ion concentration within the plant body seemed to be reflected by the active and selective ion uptake by roots and the transpiration stream. Furthermore, salt accumulation in the surface-irrigated field largely depended on the upward transport of water and ions in the soil profile affected by root absorption capacity. The information obtained in this study will contribute to the development of scientific methods for sustainable and effective plant production in irrigated fields.     

Negative conductance caused by entry of sodium and cesium ions into the potassium channels of squid axons

Internal Cs⁺, Na⁺, Li⁺, and, to a lesser degree, Rb⁺ interfere with outward current through the K pores in voltage clamped squid axons. Addition of 100 mM NaF to the perfusion medium cuts outward current for large depolarizations about in half, and causes negative conductance over a range of membrane voltages. For example, suddenly reducing membrane potential from +100 to +60 mv increases the magnitude of the outward current. Internal Cs⁺ and, to a small extent, Li⁺, also cause negative conductance. Na⁺ ions permeate at least 17 times less well through the K pores than K⁺, and Cs⁺ does not permeate measurably. The results strongly suggest that K pores have a wide and not very selective inner mouth, which accepts K⁺, Na⁺, Li⁺, Cs⁺, tetraethylammonium ion (TEA⁺), and other ions. The diameter of the mouth must be at least 8 A, which is the diameter of a TEA⁺ ion. K⁺ ions in the mouths probably have full hydration shells. The remainder of the pore is postulated to be 2.6–3.0 A in diameter, large enough for K⁺ and Rb⁺ but too small for Cs⁺ and TEA⁺. We postulate that Na⁺ ions do not enter the narrower part of the pore because they are too small to fit well in the coordination cages provided by the pore as replacements for the water molecules surrounding an ion.     

Cellular functions of a cell in a metastable equilibrium state

A unifying hypothesis which might replace some of the many ion pumps which are invoked to describe distribution of ions across living cell membranes is developed quantitatively. Resting cells are assumed to be in a metastable state such that ions are in equilibrium between an extracellular aqueous phase, in which water has the properties of the bulk liquid, and an intracellular aqueous phase in which water has enhanced structure and strongly modified solvent properties. Partition coefficients or medium effects for Na⁺, K⁺ and Cl⁻ are calculated for several cell types. It is shown that in such a hypothetical cell, possessing no ion pumps there is an amplified Donnan potential between the two phases, its sign determined by the net charge on intracellular proteins, and its magnitude increased by a separation of ions induced by the difference in solvent properties of the water in the two phases. It is shown that a cell in such a metastable state is excitable and can generate an action potential with an inward surge of Na+ followed by an outward surge of K+. An explanation is offered for the transient release of Caa+ from the sarcoplasmic reticulum following excitation of a muscle fibre. Regulation of cellular volume is shown to be a necessary result of the presence in the extracellular solution of a high concentration of Na+, an ion with a very low affinity for intracellular water. It is concluded that the principal cellular functions that are commonly attributed to the sodium pump are also a feature of a cell in a metastable equilibrium state.     

Simulations of the solvent structure for macromolecules. II. Structure of water solvating Na+-B-DNA at 300 K and a model for conformational transitions induced by solvent variations

The structure of water and its interaction energy with a fragment of B-DNA composed of 12 base pairs and of the corresponding 24 sugar and 22 phosphate units and Na⁺ ions (one at each phosphate group) are analyzed using Monte Carlo simulations. The sample of water molecules, at the simulated temperature of 300 K, is composed of 447 water molecules. The results are discussed either in terms of statistical analyses over the 2,000,000 simulated conformations (after equilibration) or with reference to an “average configuration.” Comparison is made to a simulation previously presented for the same system but without counterions. Isotherm at different relative humidity, hydration, and reactivity scales for different sites, the hydration number at each site, the structure of intraphosphate and interphosphate hydrogen-bonded filaments of water are reported and discussed. The stabilization of the B-conformation induced by the solvent with counterion (“ion-induced compression effect”) is analyzed on the base of the above findings. A preliminary model to predict conformational transition in DNA is presented. The analyses reported are very detailed to allow refined interpretations of spectroscopic (infrared, Raman, and nmr) and scattering (x-ray and neutron beam) data on DNA insolution.