DNA like-charge attraction and overcharging by divalent counterions in the presence of divalent co-ions

Strongly correlated electrostatics of DNA systems has drawn the interest of many groups, especially the condensation and overcharging of DNA by multivalent counterions. By adding counterions of different valencies and shapes, one can enhance or reduce DNA overcharging. In this paper, we focus on the effect of multivalent co-ions, specifically divalent co-ions such as SO\(_{4}^{2-}\). A computational experiment of DNA condensation using Monte Carlo simulation in grand canonical ensemble is carried out where the DNA system is in equilibrium with a bulk solution containing a mixture of salt of different valency of co-ions. Compared to systems with purely monovalent co-ions, the influence of divalent co-ions shows up in multiple aspects. Divalent co-ions lead to an increase of monovalent salt in the DNA condensate. Because monovalent salts mostly participate in linear screening of electrostatic interactions in the system, more monovalent salt molecules enter the condensate leads to screening out of short-range DNA–DNA like charge attraction and weaker DNA condensation free energy. The overcharging of DNA by multivalent counterions is also reduced in the presence of divalent co-ions. Strong repulsions between DNA and divalent co-ions and among divalent co-ions themselves lead to a depletion of negative ions near the DNA surface as compared to the case without divalent co-ions. At large distances, the DNA–DNA repulsive interaction is stronger in the presence of divalent co-ions, suggesting that divalent co-ions’ role is not only that of simple stronger linear screening.     

Non-specific binding of Na+ and Mg2+ to RNA determined by force spectroscopy methods

RNA duplex stability depends strongly on ionic conditions, and inside cells RNAs are exposed to both monovalent and multivalent ions. Despite recent advances, we do not have general methods to quantitatively account for the effects of monovalent and multivalent ions on RNA stability, and the thermodynamic parameters for secondary structure prediction have only been derived at 1M [Na(+)]. Here, by mechanically unfolding and folding a 20 bp RNA hairpin using optical tweezers, we study the RNA thermodynamics and kinetics at different monovalent and mixed monovalent/Mg(2+) salt conditions. We measure the unfolding and folding rupture forces and apply Kramers theory to extract accurate information about the hairpin free energy landscape under tension at a wide range of ionic conditions. We obtain non-specific corrections for the free energy of formation of the RNA hairpin and measure how the distance of the transition state to the folded state changes with force and ionic strength. We experimentally validate the Tightly Bound Ion model and obtain values for the persistence length of ssRNA. Finally, we test the approximate rule by which the non-specific binding affinity of divalent cations at a given concentration is equivalent to that of monovalent cations taken at 100-fold concentration for small molecular constructs.     

Kinetics and thermodynamics of thermal denaturation in acyl carrier protein.

The denaturation of Escherichia coli acyl carrier protein (ACP) in buffers containing both monovalent and divalent cations was followed by variable-temperature NMR and differential scanning calorimetry. Both high concentrations of monovalent salts (Na+) and moderate concentrations of divalent salts (Ca2+) raise the denaturation temperature, but calorimetry indicates that a significant increase in the enthalpy of denaturation is obtained only with the addition of a divalent salt. NMR experiments in both low ionic strength monovalent buffers and low ionic strength monovalent buffers containing calcium ions show exchange between native and denatured forms to be slow on the NMR time scale. However, in high ionic strength monovalent buffers, where the temperature of denaturation is elevated as it is in the presence of Ca2+, the transition is fast on the NMR time scale. These results suggest that monovalent and divalent cations may act to stabilize ACP in different ways. Monovalent ions may nonspecifically balance the intrinsic negative charge of this protein in a way that is similar for native, denatured, and intermediate forms. Divalent cations provide stability by binding to specific sites present only in the native state.     

Looping charged elastic rods: applications to protein-induced DNA loop formation

We analyze looping of thin charged elastic filaments under applied torques and end forces, using the solution of linear elasticity theory equations. In application to DNA, we account for its polyelectrolyte character and charge renormalization, calculating electrostatic energies stored in the loops. We argue that the standard theory of electrostatic persistence is only valid when the loop’s radius of curvature and close-contact distance are much larger than the Debye screening length. We predict that larger twist rates are required to trigger looping of charged rods as compared with neutral ones. We then analyze loop shapes formed on charged filaments of finite length, mimicking DNA looping by proteins with two DNA-binding domains. We find optimal loop shapes at different salt amounts, minimizing the sum of DNA elastic, DNA electrostatic, and protein elastic energies. We implement a simple model where intercharge repulsions do not affect the loop shape directly but can choose the energy-optimized shape from the allowed loop types. At low salt concentrations more open loops are favored due to enhanced repulsion of DNA charges, consistent with the results of computer simulations on formation of DNA loops by lac repressor. Then, we model the precise geometry of DNA binding by the lac tetramer and explore loop shapes, varying the confined DNA length and protein opening angle. The characteristics of complexes obtained, such as the total loop energy, stretching forces required to maintain its shape, and the reduction of electrostatic energy with increment of salt, are in good agreement with the outcomes of more elaborate numerical calculations for lac-repressor-induced DNA looping.     

Principles of RNA compaction: insights from the equilibrium folding pathway of the P4-P6 RNA domain in monovalent cations

Counterions are required for RNA folding, and divalent metal ions such as Mg(2+) are often critical. To dissect the role of counterions, we have compared global and local folding of wild-type and mutant variants of P4-P6 RNA derived from the Tetrahymena group I ribozyme in monovalent and in divalent metal ions. A remarkably simple picture of the folding thermodynamics emerges. The equilibrium folding pathway in monovalent ions displays two phases. In the first phase, RNA molecules that are initially in an extended conformation enforced by charge-charge repulsion are relaxed by electrostatic screening to a state with increased flexibility but without formation of long-range tertiary contacts. At higher concentrations of monovalent ions, a state that is nearly identical to the native folded state in the presence of Mg(2+) is formed, with tertiary contacts that involve base and backbone interactions but without the subset of interactions that involve specific divalent metal ion-binding sites. The folding model derived from these and previous results provides a robust framework for understanding the equilibrium and kinetic folding of RNA.     

Stretching single-stranded DNA: interplay of electrostatic, base-pairing, and base-pair stacking interactions.

Recent single-macromolecule observations revealed that the force/extension characteristics of single-stranded DNA (ssDNA) are closely related to solution ionic concentration and DNA sequence composition. To understand this, we studied the elastic property of ssDNA through the Monte Carlo implementation of a modified freely jointed chain (FJC), with electrostatic, base-pairing, and base-pair stacking interactions all incorporated. The simulated force-extension profiles for both random and designed sequences have attained quantitative agreements with the experimental data. In low-salt solution, electrostatic interaction dominates, and at low forces, the molecule can be more easily aligned than an unmodified FJC. In high-salt solution, secondary hairpin structure appears in ssDNA by the formation of base pairs between complementary bases, and external stretching causes a hairpin-coil structural transition, which is continuous for ssDNA made of random sequences. In designed sequences such as poly(dA-dT) and poly(dG-dC), the stacking potential between base pairs encourages the aggregation of base pairs into bulk hairpins and makes the hairpin-coil transition a discontinuous (first-order) process. The sensitivity of elongation to the base-pairing rule is also investigated. The comparison of modeling calculations and the experimental data suggests that the base pairing of single-stranded polynucleotide molecules tends to form a nested and independent planar hairpin structure rather than a random intersecting pattern.     

The Snakelike Chain Character of Unstructured RNA

In the absence of base-pairing and tertiary structure, ribonucleic acid (RNA) assumes a random-walk conformation, modulated by the electrostatic self-repulsion of the charged, flexible backbone. This behavior is often modeled as a Kratky-Porod “wormlike chain” (WLC) with a Barrat-Joanny scale-dependent persistence length. In this study we report measurements of the end-to-end extension of poly(U) RNA under 0.1 to 10 pN applied force and observe two distinct elastic-response regimes: a low-force, power-law regime characteristic of a chain of swollen blobs on long length scales and a high-force, salt-valence-dependent regime consistent with ion-stabilized crumpling on short length scales. This short-scale structure is additionally supported by force- and salt-dependent quantification of the RNA ion atmosphere composition, which shows that ions are liberated under stretching; the number of ions liberated increases with increasing bulk salt concentration. Both this result and the observation of two elastic-response regimes directly contradict the WLC model, which predicts a single elastic regime across all forces and, when accounting for scale-dependent persistence length, the opposite trend in ion release with salt concentration. We conclude that RNA is better described as a “snakelike chain,” characterized by smooth bending on long length scales and ion-stabilized crumpling on short length scales. In monovalent salt, these two regimes are separated by a characteristic length that scales with the Debye screening length, highlighting the determining importance of electrostatics in RNA conformation.     

The Snakelike Chain Character of Unstructured RNA

In the absence of base-pairing and tertiary structure, ribonucleic acid (RNA) assumes a random-walk conformation, modulated by the electrostatic self-repulsion of the charged, flexible backbone. This behavior is often modeled as a Kratky-Porod “wormlike chain” (WLC) with a Barrat-Joanny scale-dependent persistence length. In this study we report measurements of the end-to-end extension of poly(U) RNA under 0.1 to 10 pN applied force and observe two distinct elastic-response regimes: a low-force, power-law regime characteristic of a chain of swollen blobs on long length scales and a high-force, salt-valence-dependent regime consistent with ion-stabilized crumpling on short length scales. This short-scale structure is additionally supported by force- and salt-dependent quantification of the RNA ion atmosphere composition, which shows that ions are liberated under stretching; the number of ions liberated increases with increasing bulk salt concentration. Both this result and the observation of two elastic-response regimes directly contradict the WLC model, which predicts a single elastic regime across all forces and, when accounting for scale-dependent persistence length, the opposite trend in ion release with salt concentration. We conclude that RNA is better described as a “snakelike chain,” characterized by smooth bending on long length scales and ion-stabilized crumpling on short length scales. In monovalent salt, these two regimes are separated by a characteristic length that scales with the Debye screening length, highlighting the determining importance of electrostatics in RNA conformation.     

THE LACK OF SPECIFICITY TOWARDS SALTS IN THE ACTIVATION OF CHOLINE ACETYLTRANSFERASE FROM HUMAN PLACENTA

Abstract— The effects of monovalent and divalent anions on the choline acetyltransferase reaction have been determined at high (5.0 mM) and low (0.58 mM) choline. At 0.58 mM-choline, both monovalent and divalent anions activate the enzyme ±9 fold; however, at 5.0mM-choline, monovalent anions activate the enzyme ±25 fold, while divalent anions activate ±9 fold. Both monovalent and divalent anions show uncompetitive activation with respect to choline. When either dimethylaminoethanol, N -(2-hydroxyethyl)- N -methyl piperidinium iodide, or N -(2-hydroxyethyl)- N -propyl pyrrolidinium iodide was substituted for choline, activation by monovalent or divalent anions was only 2.5-4 fold. With AcCoA as substrate the ChA reaction can be increased ±20 fold by increased salts; however, with acetyl dephosphoCoA as substrate, the reaction is insensitive to the salt concentration. Similar salt effects on the ChA reaction, as measured in the direction of acetylcholine synthesis, have been demonstrated in the reverse reaction. In addition, inhibition of the forward reaction by acetylcholine has been measured as a function of sodium chloride concentration. Although the K₁ for acetylcholine increases with increasing salt, this change in K ₁, parallels the increase in the K _m for choline. These results support the hypothesis that both monovalent and divalent anions activate choline acetyltransferase by the same singular mechanism; which is to increase the rate of dissociation of coenzyme A from the enzyme.     

Cloud-point temperatures for lysozyme in electrolyte solutions: effect of salt type, salt concentration and pH

Liquid-liquid phase-separation data were obtained for aqueous saline solutions of hen egg-white lysozyme at a fixed protein concentration (87 g/l). The cloud-point temperature (CPT) was measured as a function of salt type and salt concentration to 3 M, at pH 4.0 and 7.0. Salts used included those from mono and divalent cations and anions. For the monovalent cations studied, as salt concentration increases, the CPT increases. For divalent cations, as salt concentration rises, a maximum in the CPT is observed and attributed to ion binding to the protein surface and subsequent water structuring. Trends for sulfate salts were dramatically different from those for other salts because sulfate ion is strongly hydrated and excluded from the lysozyme surface. For anions at fixed salt concentration, the CPT decreases with rising anion kosmotropic character. Comparison of CPTs for pH 4.0 and 7.0 revealed two trends. At low ionic strength for a given salt, differences in CPT can be explained in terms of repulsive electrostatic interactions between protein molecules, while at higher ionic strength, differences can be attributed to hydration forces. A model is proposed for the correlation and prediction of the CPT as a function of salt type and salt concentration. NaCl was chosen as a reference salt, and CPT deviations from that of NaCl were attributed to hydration forces. The Random Phase Approximation, in conjunction with a square-well potential, was used to calculate the strength of protein-protein interactions as a function of solution conditions for all salts studied.     

Excessive counterion condensation on immobilized ssDNA in solutions of high ionic strength

We present experiments on the bias-induced release of immobilized, single-stranded (ss) 24-mer oligonucleotides from Au-surfaces into electrolyte solutions of varying ionic strength. Desorption is evidenced by fluorescence measurements of dye-labeled ssDNA. Electrostatic interactions between adsorbed ssDNA and the Au-surface are investigated with respect to 1), a variation of the bias potential applied to the Au-electrode; and 2), the screening effect of the electrolyte solution. For the latter, the concentration of monovalent salt in solution is varied from 3 to 1600 mM. We find that the strength of electric interaction is predominantly determined by the effective charge of the ssDNA itself and that the release of DNA mainly occurs before the electrochemical double layer has been established at the electrolyte/Au interface. In agreement with Manning's condensation theory, the measured desorption efficiency (etarel) stays constant over a wide range of salt concentrations; however, as the Debye length is reduced below a value comparable to the axial charge spacing of the DNA, etarel decreases substantially. We assign this effect to excessive counterion condensation on the DNA in solutions of high ionic strength. In addition, the relative translational diffusion coefficient of ssDNA in solution is evaluated for different salt concentrations.     

Electron and scanning force microscopy studies of alterations in supercoiled DNA tertiary structure.

The configuration of supercoiled DNA (scDNA) was investigated by electron microscopy and scanning force microscopy. Changes in configuration were induced by varying monovalent/divalent salt concentrations and manifested by variation in the number of nodes (crossings of double helical segments). A decrease in the concentration of monovalent cations from 50 mM to approximately 1 mM resulted in a significant change of apparent configuration of negatively supercoiled DNA from a plectonemic form with virtually approximately 15 nodes (the value expected for molecules of approximately 3000 bp) to one or two nodes. This result was in good agreement with values calculated using an elastic rod model of DNA and salt concentration in the range of 5-50 mM. The effect did not depend on the identity of the monovalent cation (Na(+), K(+)) or the nature of the support used for electron microscopy imaging (glow-discharged carbon film, polylysine film). At very low salt concentrations, a single denatured region several hundred base-pairs in length was often detected. Similarly, at low concentrations of divalent cations (Mg(2+), Ca(2+), Zn(2+)), scDNA was apparently relaxed, although the effect was slightly dependent on the nature of the cation. Positively supercoiled DNA behaved in a manner different from that of its negative counterpart when the ion concentration was varied. As expected for these molecules, an increase in salt concentration resulted in an apparent relaxation; however, a decrease in salt concentration also led to an apparent relaxation manifested by a slight decrease in the number of nodes. Scanning force microscopy imaging of negatively scDNA molecules deposited onto a mica surface under various salt conditions also revealed an apparent relaxation of scDNA molecules. However, due to weak interactions with the mica surface in the presence of a mixture of mono/divalent cations, the effect occurred under conditions differing from those used for electron microscopy. We conclude that the observed changes in scDNA configuration are inherent to the DNA structure and do not reflect artifacts arising from the method(s) of sample preparation.     

Electrostatic effects on the stability of condensed DNA in the presence of divalent cations.

Cylindrical cell model Poisson-Boltzmann (P-B) calculations are used to evaluate the electrostatic contributions to the relative stability of various DNA conformations (A, B, C, Z, and single-stranded (ss) with charge spacings of 3.38 and 4.2 A) as a function of interhelix distance in a concentrated solution of divalent cations. The divalent ion concentration was set at 100 mM, to compare with our earlier reports of spectroscopic and calorimetric experiments, which demonstrate substantial disruption of B-DNA geometry. Monovalent cations neutralize the DNA phosphates in two ways, corresponding to different experimental situations: 1) There is no significant contribution to the ionic strength from the neutralizing cations, corresponding to DNA condensation from dilute solution and to osmotic stress experiments in which DNA segments are brought into close proximity to each other in the presence of a large excess of buffer. 2) The solution is uniformly concentrated in DNA, so that the neutralizing cations add significantly to those in the buffer at close DNA packing. In case 1), conformations with lower charge density (Z and ssDNA) have markedly lower electrostatic free energies than B-DNA as the DNA molecules approach closely, due largely to ionic entropy. If the divalent cations bind preferentially to single-stranded DNA or a distorted form of B-DNA, as is the case with transition metals, the base pairing and stacking free energies that stabilize the double helix against electrostatic denaturation may be overcome. Strong binding to the bases is favored by the high concentration of divalent cations at the DNA surface arising from the large negative surface potential; the surface concentration increases sharply as the interhelical distance decreases. In case 2), the concentration of neutralizing monovalent cations becomes very large and the electrostatic free energy difference between secondary structures becomes small as the interhelical spacing decreases. Such high ionic concentrations will be expected to modify the stability of DNA by changing water activity as well as by screening electrostatic interactions. This may be the root of the decreased thermal stability of DNA in the presence of high concentrations of magnesium ions.     

Conformation of ring single-stranded DNA measured by DNA origami structures

Measuring the mechanical properties of single-stranded DNA (ssDNA) is a complex challenge that has been addressed lately by different methods. We measured the persistence length of ring ssDNA using a combination of a special DNA origami structure, a self-avoiding ring polymer simulation model, and nonparametric estimation statistics. The method overcomes the complexities set forth by previously used methods. We designed the DNA origami nano structures and measured the ring ssDNA polymer conformations using atomic force microscopy. We then calculated their radius of gyration, which was used as a fitting parameter for finding the persistence length. As there is no simple formulation for the radius of gyration distribution, we developed a simulation program consisting of a self-avoiding ring polymer to fit the persistence length to the experimental data. ssDNA naturally forms stem-loops, which should be taken into account in fitting a model to the experimental measurement. To overcome that hurdle, we found the possible loops using minimal energy considerations and used them in our fitting procedure of the persistence length. Due to the statistical nature of the loops formation, we calculated the persistence length for different percentages of loops that are formed. In the range of 25–75% loop formation, we found the persistence length to be 1.9–4.4 nm, and for 50% loop formation we get a persistence length of 2.83 ± 0.63 nm. This estimation narrows the previously known persistence length and provides tools for finding the conformations of ssDNA.     

Structure and Conformation of Wild-Type Bacterial Lipopolysaccharide Layers at Air-Water Interfaces

The outer membrane of Gram-negative bacteria is of great scientific interest because it mediates the action of antimicrobial agents. The membrane surface is composed of lipopolysaccharide (LPS) molecules with negatively charged oligosaccharide headgroups. To a certain fraction, LPSs additionally display linear polysaccharides termed O-side chains (OSCs). Structural studies on bacterial outer surfaces models, based on LPS monolayers at air-water interfaces, have so far dealt only with rough mutant LPSs lacking these OSCs. Here, we characterize monolayers of wild-type LPS from Escherichia coli O55:B5 featuring strain-specific OSCs in the presence of defined concentrations of monovalent and divalent ions. Pressure-area isotherms yield insight into in-plane molecular interactions and monolayer elastic moduli. Structural investigations by x-ray and neutron reflectometry reveal the saccharide conformation and allow quantifying the area per molecule and the fraction of LPS molecules carrying OSCs. The OSC conformation is satisfactorily described by the self-consistent field theory for end-grafted polymer brushes. The monolayers exhibit a significant structural response to divalent cations, which goes beyond generic electrostatic screening.     

Effects of neutral salts on the conformational state of glycogen, interacting with iodine

The effects of salts on helix formation of linear (outer) chains of branched alpha-glucane, muscle glycogen, was judged upon by the state of polyiodine chromophore of the iodine reaction surrounded by a polysaccharide asymmetric helix and recorded by the circular dichroism method. It was demonstrated that apart from the known changes in the absorption of the iodine-glycogen complex the salts induce changes in the ellipticity by affecting the helix formation of the linear chains of the polysaccharide. The nature of these effects depends on the type and concentration of the salt used. Monovalent metal salts produce a more favourable effect on helix formation than the divalent metal salts. Among divalent metal salts transient metal salts produce a weaker effect. The action of neutral salts on helix formation is of dual nature: helix formation and helix destruction. The helix formation is largely impaired by high concentrations of LiCl (greater than 6 M) or CaCl2 (greater than 2M). It was shown that a certain role in the mechanism of neutral salt action on helix formation belongs both to electrostatic and lyotropic effects. The latter determine the effects of salts on helix formation at concentrations above the monomolar one, when the specific effect of a salt is especially well-pronounced. The salts which enhance the orderliness of H2O structure produce a favourable effect on helix formation of the linear polysaccharide chains and, consequently, on the iodine reaction. In terms of molar efficiency of their action on the iodine reaction the salts correspond to the Hofmeister lyotropic sequence.     

Determination of the elastic properties of short ssDNA molecules by mechanically folding and unfolding DNA hairpins

The characterization of elastic properties of biopolymers is crucial to understand many molecular reactions determined by conformational bending fluctuations of the polymer. Direct measurement of such elastic properties using single‐molecule methods is usually hindered by the intrinsic tendency of such biopolymers to form high‐order molecular structures. For example, single‐stranded deoxyribonucleic acids (ssDNA) tend to form secondary structures such as local double helices that prevent the direct measurement of the ideal elastic response of the ssDNA. In this work, we show how to extract the ideal elastic response in the entropic regime of short ssDNA molecules by mechanically pulling two‐state DNA hairpins of different contour lengths. This is achieved by measuring the force dependence of the molecular extension and stiffness on mechanically folding and unfolding the DNA hairpin. Both quantities are fit to the worm‐like chain elastic model giving values for the persistence length and the interphosphate distance. This method can be used to unravel the elastic properties of short ssDNA and RNA sequences and, more generally, any biopolymer that can exhibit a cooperative two‐state transition between mechanically folded and unfolded states (such as proteins). © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1193–1199, 2014.     

两价盐对C_8－卵磷脂微团溶液液－液相分离的影响

采用激光散射等方法测定了在添加不同的两价无机盐情况下,Ｃ８－卵磷脂微团溶液的液－液相分离曲线,及其相变临界温度随盐类型和盐离子强度的变化。并从理论上分析了两价盐对Ｃ８－卵磷脂微团溶液吉布斯自由能的影响,推导出－关于盐对该微团溶液相交临界温度影响的半经验半理论公式,可满意地描述该微团溶液的液－液相分离受益调控的规律。     

Electrostatic effects in short superhelical DNA

We present Monte Carlo simulations of the equilibrium configurations of short closed circular DNA that obeys a combined elastic, hard-sphere, and electrostatic energy potential. We employ a B-spline representation to model chain configuration and simulate the effects of salt on chain folding by varying the Debye screening parameter. We obtain global equilibrium configurations of closed circular DNA, with several imposed linking number differences, at two salt concentrations (specifically at the extremes of no added salt and the high salt regime), and for different chain lengths. Minimization of the composite elastic/long-range potential energy under the constraints of ring closure and fixed chain length is found to produce structures that are consistent with the configurations of short supercoiled DNA observed experimentally. The structures generated under the constraints of an electrostatic potential are less compact than those subjected only to an elastic term and a hard-sphere constraint. For a fixed linking number difference greater than a critical value, the interwound structures obtained under the condition of high salt are more compact than those obtained under the condition of no added salt. In the case of no added salt, the electrostatic energy plays a dominant role over the elastic energy in dictating the shape of the closed circular DNA. The DNA supercoil opens up with increasing chain length at low salt concentration. A branched three-leaf rose structure with a fixed linking number difference is higher in energy than the interwound form at both salt concentrations employed here.