DNA Charge Neutralization Research Articles

DNA-Lysozyme Nanoarchitectonics: Quantitative Investigation on Charge Inversion and Compaction.

The interaction between DNA and proteins is fundamentally important not only for basic research in biology, but also for potential applications in nanotechnology. In the present study, the complexes formed by λ DNA and lysozyme in a dilute aqueous solution have been investigated using magnetic tweezers (MT), dynamic light scattering (DLS), and atomic force microscopy (AFM). We found that lysozyme induced DNA charge inversion by measuring its electrophoretic mobility by DLS. Lysozyme is very effective at neutralizing the positive charge of DNA, and its critical charge ration to induce charge inversion in solution is only 2.26. We infer that the high efficiency of charge neutralization is due to the highly positively charged (+8 e) and compact structure of lysozyme. When increasing the concentration of lysozymes from 6 ng·µL−1 to 70 ng·µL−1, DNA mobility (at fixed concentration of 2 ng·µL−1) increases from −2.8 to 1.5 (in unit of 10−4 cm2·V−1·S), implying that the effective charge of DNA switches its sign from negative to positive in the process. The corresponding condensing force increased from 0 pN to its maximal value of about 10.7 pN at concentrations of lysozyme at 25 ng·µL−1, then decreases gradually to 3.8 pN at 200 ng·µL−1. The maximal condensing force occurs at the complete DNA charge neutralization point. The corresponding morphology of DNA–lysozyme complex changes from loosely extensible chains to compact globule, and finally to less compact flower-like structure due to the change of attached lysozyme particles as observed by AFM.

The Modulation of Chitosan-DNA Interaction by Concentration and pH in Solution.

Chitosan has been widely used to prepare a DNA carrier for highly efficient and non-toxic gene therapy. In the present study, we investigated DNA charge neutralization and compaction by chitosan in solutions of various pH levels by dynamic light scattering (DLS), magnetic tweezers (MT), and atomic force microscopy (AFM). We found that when chitosan concentration is higher than a critical value (0.2 µM), corresponding the ratio of phosphate and NH2 in chitosan , the electrophoretic mobility of DNA-chitosan complex maintains an almost constant value when pH of solution is less 6.5, the isoelectric point of chitosan. Then it decreases with increasing pH of solution. However, when chitosan concentration is lower than the critical value, the mobility of the complex increases with pH in the range of acidity and reaches the maximum when the pH of the solution approaches the isoelectric point of chitosan. It finally decreases with increasing pH in solutions. The corresponding condensing force of the DNA-chitosan complex measured by single molecular MT changes accordingly with its charge neutralization in the same solution concentration (20 µM) and is consistent with the DLS measurements. This phenomenon might be related to the weakening interaction between DNA and chitosan in low pH solutions, and is verified by measuring the ratio of free chitosan to DNA complex in solutions. We also observed the various morphologies of DNA-chitosan complexes, such as ring, rod, flower, braid, and other structures, under different degrees of deacetylation, molecular weight, solution concentration and pH in solutions by AFM.

DNA Compaction and Charge Neutralization Regulated by Divalent Ions in very Low pH Solution.

DNA conformation is strongly dependent on the valence of counterions in solution, and a valence of at least three is needed for DNA compaction. Recently, we directly demonstrated DNA compaction and its regulation, mediated by divalent cations, by lowering the pH of a solution. In the present study, we found that the critical electrophoretic mobility of DNA is promoted to around −1.0 × 10−4 cm2 V−1 s−1 to incur DNA compaction or condensation in a tri- and tetravalent counterions solution, corresponding to an about 89% neutralized charge fraction of DNA. This is also valid for DNA compaction by divalent counterions in a low pH solution. It is notable that the critical charge neutralization of DNA for compaction is only about 1% higher than the saturated charge fraction of DNA in a mild divalent ion solution. We also found that DNA compaction by divalent cations at low pH is weakened and even decondensed with an increasing concentration of counterions.

The promotion and suppression of DNA charge neutralization by the cosolute ectoine.

Ectoine, a cosolute and osmolyte, is used by extremophilic microorganisms to maintain an osmotic equilibrium of cells with their surrounding medium under conditions of extreme salinity or thermal and pressure stresses. It is also considered a protectant of biomolecules such as protein and DNA in cells. In the present study, we investigate its influence on DNA charge neutralization and compaction through dynamic light scattering (DLS), atomic force microscopy (AFM) and single molecular magnetic tweezers (MT). We found that ectoine can promote DNA charge neutralization induced by multivalent cations at mild cosolute concentration in solution. When the concentration of ectoine is high enough, however, a mixed effect of promotion and suppression can be found under the same ionic conditions. In this case, the electrophoretic mobility (EM) of DNA is promoted in the region of low cation concentration, while suppressed in the region of high counterionic concentration. The charge neutralization of DNA by ectoine is also related to DNA compaction. The promotion and suppression of DNA compaction by ectoine was observed by AFM imaging. The condensed structure of DNA becomes more compact and then loose once more with the increasing concentration of ectoine. Meanwhile, the condensing forces of DNA measured by magnetic tweezers shows the same trend as does the DNA EM. We explained the experimental findings through the combined effect of two intrinsic features of ectoine, preferential exclusion and enhancement of the dielectric constant of the medium.

The Mixing Counterion Effect on DNA Compaction and Charge Neutralization at Low Ionic Strength.

DNA compaction and charge neutralization in a mixing counterion solution involves competitive and cooperative electrostatic binding, and sometimes counterion complexation. At normal ionic strength, it has been found that the charge neutralization of DNA by the multivalent counterion is suppressed when being added extra mono- and di-valent counterions. Here, we explore the effect mixing counterion on DNA compaction and charge neutralization under the condition of low ionic strength. Being quite different from normal ionic strength, the electrophoretic mobility of DNA in multivalent counterion solution (octalysine, spermine) increases the presence of mono- and di-valent cations, such as sodium and magnesium ions. It means that the charge neutralization of DNA by the multivalent counterion is promoted rather than suppressed when introducing extra mono- and di-valent counterions into solution. This conclusion is also supported by the measurement of condensing and unraveling forces of DNA condensates under the same condition by single molecular magnetic tweezers. This mixing effect can be attributed to the cooperative electrostatic binding of counterions to DNA when the concentration of counterions in solution is below a critical concentration.

Membrane-Spanning DNA Ion Channels: Conductance Mechanism, Electro-Osmotic Transport and Mechanical Gating

DNA self-assembly has emerged as a new paradigm for design of biomimetic ion channels and transporters. Several experimental groups have already reported spontaneous insertion of DNA constructs into lipid bilayer membranes and measurements of ionic flux through the channels. However, the structure such DNA ion channels in the lipid bilayer membrane environment and their conductance mechanisms have remained undetermined. Two years ago, we reported the first atomistic molecular dynamics (MD) simulations of DNA origami objects, characterizing their chemical and mechanical properties in unprecedented detail [PNAS, 110(50), 20099 (2013)]. Here, we extend our MD method to characterize the biophysical properties of DNA ion channels embedded in lipid bilayer membranes. First, we investigate the effect of two popular methods of DNA construct insertion—partial neutralization of DNA charge and hydrophobic anchoring—on the structure of the surrounding lipid bilayer. Although our simulations confirm that both methods yield stable assembly, profound differences are found in the structure of the DNA—lipid interface. Second, we find that the transmembrane ionic current flows not only through the central pore of the channel, but also around the walls of the channel and through the gaps in the DNA structure. The ratio of the current through each path depends on the design of the channel and the lipid—DNA interface. Surprisingly, we find the conductance of a DNA channel to depend on the membrane tension, making them potentially suitable for force-sensing applications. Finally, we show that electro-osmosis governs the transport of drug-like molecules through the DNA ion channels, suggesting possible applications of such systems in biosensing and drug delivery.

The effect of pH on charge inversion and condensation of DNA.

Charge inversion and condensation of DNA in solutions of trivalent and quadrivalent counterions are significantly influenced by the pH value of the solution. We systematically investigated the condensation and charge compensation of DNA by spermidine, hexammine cobalt(iii) (cohex, [Co(NH3)6](3+)) and spermine in solutions of a wide range of pH values from 3 to 9.3 by dynamic light scattering, magnetic tweezers, and atomic force microscopy. In trivalent counterion solution, we found that there is a critical concentration (0.75 mM for cohex and 0.5 mM for spermidine), under which the electrophoresis mobility of DNA initially increases, reaches a maximum, and finally decreases when the pH value is decreased. In contrast, above the critical concentration, the electrophoretic mobility of DNA increases monotonously with decreasing pH value of the solution. The corresponding condensing force has the same dependence on the pH value. However, for the case of quadrivalent counterions, the electrophoretic mobility of DNA is monotonously promoted by lowering the pH value of the solution at any concentration of counterions in which charge inversion of DNA may occur. In atomic force microscopy images and force spectroscopy of magnetic tweezers, we found that maximal charge neutralization and condensation force correspond to the most compact DNA condensation. We propose a mechanism of promoting DNA charge neutralization: small and highly mobile hydrogen ions tend to attach to the DNA-counterion complex to further neutralize its remaining charge, which is related to the surface area of the complex. Therefore, this further neutralization is prominent when the complex is toroidal which corresponds to the case of mild ion concentration while it is less prominent for more compact globules or rod complexes at high counterion concentration.

CHARMM Drude Polarizable MD Simulations Reproduce Solution X-Ray Diffraction Patterns for B-DNA Sequences and Predict Differential Impact of the Li+, Na+, K+ and Rb+ Ions on DNA Conformational Properties

Recently we have presented the first generation CHARMM Drude polarizable force field for DNA, which is capable of reproducing the main conformational features of DNA in solution, such as A-to-B equilibrium and transitions between BI and BII substates. Our current efforts are directed towards further model improvement, by achieving a proper balance of the interactions among surrounding mobile ions, water and DNA. Compared to the additive (non-polarizable) models, explicit treatment of the electronic polarizability in the Drude model leads to a markedly improved description of the interplay between the ionic atmosphere and DNA conformational behavior. In particular, the Drude model is shown to more accurately reproduce counterion condensation theory predictions of DNA charge neutralization by condensed ions, as well as the experimental data on the competitive binding of Li+, Na+, K+ and Rb+ ions to DNA. The most intriguing results is that the model predicts a differential impact of these seemingly similar monovalent cations on DNA conformational properties - a phenomenon not observed in the state-of-the-art CHARMM36 and AMBER atomistic additive models. In addition, the Drude model reproduces the solution X-ray diffraction patterns for a number of B form DNA sequences at a level of accuracy similar to, or exceeding that of the above mentioned additive models. The obtained results indicate that CHARMM Drude polarizable MD simulations provide a more realistic model of the physical forces involved in the interactions of DNA with its ionic environment, offering the potential to yield new insights into salt-mediated biological processes involving DNA, such as protein-DNA recognition and chromatin folding.

Balancing the interactions of ions, water, and DNA in the Drude polarizable force field.

Recently we presented a first-generation all-atom Drude polarizable force field for DNA based on the classical Drude oscillator model, focusing on optimization of key dihedral angles followed by extensive validation of the force field parameters. Presently, we describe the procedure for balancing the electrostatic interactions between ions, water, and DNA as required for development of the Drude force field for DNA. The proper balance of these interactions is shown to impact DNA stability and subtler conformational properties, including the conformational equilibrium between the BI and BII states, and the A and B forms of DNA. The parametrization efforts were simultaneously guided by gas-phase quantum mechanics (QM) data on small model compounds and condensed-phase experimental data on the hydration and osmotic properties of biologically relevant ions and their solutions, as well as theoretical predictions for ionic distribution around DNA oligomer. In addition, fine-tuning of the internal base parameters was performed to obtain the final DNA model. Notably, the Drude model is shown to more accurately reproduce counterion condensation theory predictions of DNA charge neutralization by the condensed ions as compared to the CHARMM36 additive DNA force field, indicating an improved physical description of the forces dictating the ionic solvation of DNA due to the explicit treatment of electronic polarizability. In combination with the polarizable DNA force field, the availability of Drude polarizable parameters for proteins, lipids, and carbohydrates will allow for simulation studies of heterogeneous biological systems.

Interaction of new butyltin citrate complex with lipid model membrane and DNA

Organotin(IV) complexes show a wide variety of biological activity: bactericidal, fungicidal, caricidal, and pesticidal. Many of them are more effective than traditional heavy metal anticancer drugs. The aim of the present study was to investigate the interaction of a new dibutyltin complex with citric acid (DBTC) with lipid membrane and plasmid DNA. The effect of this compound on the multilamellar liposomes formed with dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, and dimyristoylphosphatidylethanolamine was studied mainly by the means of differential scanning calorimetry and additionally by the steady-state fluorimetry. Calorimetrical results together with fluorescence spectroscopy suggest that investigated complex interacts with lipids and probably locates itself in the hydrophilic part of the membrane. The effect of DBCT on plasmid DNA was investigated using single-molecule florescence technique and time-correlated single photon counting fluorescence correlation spectroscopy. We could conclude then that DBTC–DNA interaction occurs due to the interaction with the DNA phosphate group and charge neutralization which then leads to DNA compaction.

Ion Competition in Condensed DNA Arrays in the Attractive Regime

Physical origin of DNA condensation by multivalent cations remains unsettled. Here, we report quantitative studies of how one DNA-condensing ion (Cobalt3+ Hexammine, or Co3+Hex) and one nonDNA-condensing ion (Mg2+) compete within the interstitial space in spontaneously condensed DNA arrays. As the ion concentrations in the bath solution are systematically varied, the ion contents and DNA-DNA spacings of the DNA arrays are determined by atomic emission spectroscopy and x-ray diffraction, respectively. To gain quantitative insights, we first compare the experimentally determined ion contents with predictions from exact numerical calculations based on nonlinear Poisson-Boltzmann equations. Such calculations are shown to significantly underestimate the number of Co3+Hex ions, consistent with the deficiencies of nonlinear Poisson-Boltzmann approaches in describing multivalent cations. Upon increasing the concentration of Mg2+, the Co3+Hex-condensed DNA array expands and eventually redissolves as a result of ion competition weakening DNA-DNA attraction. Although the DNA-DNA spacing depends on both Mg2+ and Co3+Hex concentrations in the bath solution, it is observed that the spacing is largely determined by a single parameter of the DNA array, the fraction of DNA charges neutralized by Co3+Hex. It is also observed that only ∼20% DNA charge neutralization by Co3+Hex is necessary for spontaneous DNA condensation. We then show that the bath ion conditions can be reduced to one variable with a simplistic ion binding model, which is able to describe the variations of both ion contents and DNA-DNA spacings reasonably well. Finally, we discuss the implications on the nature of interstitial ions and cation-mediated DNA-DNA interactions.

Detection of DNA hybridization by field-effect DNA-based biosensors: mechanisms of signal generation and open questions

We model theoretically the electrostatic effects taking place upon DNA hybridization in dense DNA arrays immobilized on a layer of Au nano-particles deposited on the surface of a field-effect-based DNA capacitive biosensor. We consider the influence of separation of a charged analyte from the sensor surface and the salinity of electrolyte solution, in the framework of both linear and nonlinear Poisson–Boltzmann theories. The latter predicts a substantially weaker sensor signals due to electrostatic saturation effects that is the main conclusion of this paper. We analyze how different physical parameters of dense DNA brushes affect the magnitude of hybridization signals. The list includes the fraction of DNA charge neutralization, the length and spatial conformations of adsorbed DNA molecules, as well as the discreteness of DNA charges. We also examine the effect of Donnan ionic equilibrium in DNA lattices on the sensor response. The validity of theoretical models is contrasted against recent experimental observations on detection of DNA hybridization via its intrinsic electric charge. The sensitivity of such biochemical sensing devices, their detection limit, and DNA hybridization efficiency are briefly discussed in the end.

DNA condensation in bacteria: Interplay between macromolecular crowding and nucleoid proteins

The volume of a typical Eschericia coli nucleoid is roughly 10 4 times smaller than the volume of a freely coiling linear DNA molecule with the same length as the E. coli genome. We review the main forces that have been suggested to contribute to this compaction factor: macromolecular crowding (that “pushes” the DNA together), DNA charge neutralization by various polycationic species (that “glues” the DNA together), and finally, DNA deformations due to DNA supercoiling and nucleoid proteins. The direct contributions of DNA supercoiling and nucleoid proteins to the total compaction factor are probably small. Instead, we argue that the formation of the bacterial nucleoid can be described as a consequence of the influence of macromolecular crowding on thick, supercoiled protein-DNA fibers, that have been partly charge neutralized by small multivalent cations.

Knockout of the gene encoding the kinetoplast-associated protein 3 (KAP3) in Trypanosoma cruzi: Effect on kinetoplast organization, cell proliferation and differentiation

Kinetoplast DNA (kDNA) of trypanosomatid protozoa consists of an unusual arrangement of two types of circular molecules catenated into a single network: (1) a few maxicircles that encode various mitochondrial enzyme subunits and rRNA in a cryptic pattern and (2) thousands of minicircles that encode guide RNAs (gRNAs). kDNA is associated with proteins, known as kinetoplast-associated proteins (KAPs), which condense the kDNA network. However, little is known about the KAPs of Trypanosoma cruzi, a parasite that displays different kDNA condensation patterns during its complex morphogenetic development. We cloned the T. cruzi gene encoding TcKAP3 (kinetoplast-associated protein 3). TcKAP3 is a single-copy gene that is transcribed into a 1.8-kb mRNA molecule and expressed in all stages of the parasite. Mouse antiserum raised against recombinant TcKPA3 recognized a 21.8kDa protein, which was found, by indirect immunofluorescence and immunoelectron microscopy, to be associated with the T. cruzi kinetoplast. Several features of TcKAP3, such as its small size, basic nature and similarity with KAP3 from the insect trypanosomatid Crithidia fasciculata, are consistent with a role in DNA charge neutralization and condensation. This suggests that this protein is involved in organizing the kDNA network. Gene deletion was used to investigate TcKAP3 function. Here we investigated the T. cruzi KAP3 null mutant, analyzing its fitness during proliferation, differentiation and infectivity.

Expression and subcellular localization of kinetoplast-associated proteins in the different developmental stages of Trypanosoma cruzi

BackgroundThe kinetoplast DNA (kDNA) of trypanosomatids consists of an unusual arrangement of circular molecules catenated into a single network. The diameter of the isolated kDNA network is similar to that of the entire cell. However, within the kinetoplast matrix, the kDNA is highly condensed. Studies in Crithidia fasciculata showed that kinetoplast-associated proteins (KAPs) are capable of condensing the kDNA network. However, little is known about the KAPs of Trypanosoma cruzi, a parasitic protozoon that shows distinct patterns of kDNA condensation during their complex morphogenetic development. In epimastigotes and amastigotes (replicating forms) the kDNA fibers are tightly packed into a disk-shaped kinetoplast, whereas trypomastigotes (non-replicating) present a more relaxed kDNA organization contained within a rounded structure. It is still unclear how the compact kinetoplast disk of epimastigotes is converted into a globular structure in the infective trypomastigotes.ResultsIn this work, we have analyzed KAP coding genes in trypanosomatid genomes and cloned and expressed two kinetoplast-associated proteins in T. cruzi: TcKAP4 and TcKAP6. Such small basic proteins are expressed in all developmental stages of the parasite, although present a differential distribution within the kinetoplasts of epimastigote, amastigote and trypomastigote forms.ConclusionSeveral features of TcKAPs, such as their small size, basic nature and similarity with KAPs of C. fasciculata, are consistent with a role in DNA charge neutralization and condensation. Additionally, the differential distribution of KAPs in the kinetoplasts of distinct developmental stages of the parasite, indicate that the kDNA rearrangement that takes place during the T. cruzi differentiation process is accompanied by TcKAPs redistribution.

Divalent cation distribution in dinoflagellate chromosomes imaged by high-resolution ion probe mass spectrometry

From a variety of analytical electron microscopy experiments, the chromosomes of dinoflagellates are known to contain sizeable amounts of cations, the latter thought to contribute to the neutralization of the negative charge carried by the phosphate groups in the DNA backbone. From previous Ca and Mg chelation experiments, it is also known that these cations are necessary for the compaction and preservation of the chromosome architecture. Similar conclusions have been recently presented by our group concerning mammalian mitotic chromosomes, in studies based on secondary ion mass spectrometry (SIMS) carried out with the University of Chicago high-resolution scanning ion microprobe (UC-SIM). We have now applied this instrument to image the distribution of DNA-bound Ca 2+ and Mg 2+ in dinoflagellate chromosomes, a goal that could not be attained earlier by analytical electron microscopy. Analyzed quantitatively and imaged here by SIMS for the first time, through their cation content, are the chromosomes of the dinoflagellates Prorocentrum micans, Gymnodinium mikimotoi and Gymnodinium dorsum. The cell nuclei were isolated and prepared for SIMS analysis with a minimal protocol (mechanical fractionation in culture medium followed by ethanol drying), which did not expose the samples to artifact-creating, alien chemical agents. By this approach, we have confirmed the earlier findings by several authors, and contributed new structural information provided by our ion probe capability to erode the sample surface layer by layer (SIMS tomography). Dinoflagellates, due to the absence of histones, represent an ideal model system where cations may bind directly with DNA, allowing comparisons to be made with recently reported X-ray crystallography results at atomic resolution. Such comparisons yielded quantitative confirmation that the Ca 2++Mg 2+ concentrations found for e.g. P. micans are consistent with those anticipated to provide complete charge neutralization of naked DNA by cations, also resulting in maximal DNA compaction.

DNA charge neutralization by linear polymers. II. Reversible binding

We model the way in which polymers bind to DNA and neutralize its charged backbone by analyzing the dynamics of the distribution of gaps along the DNA. We generalize existing theory for irreversible binding to construct deterministic models which include polymer removal, movement along the DNA, and allow for binding with overlaps. We show that reversible binding alters the capacity of the DNA for polymers by allowing the rearrangement of polymer positions over a longer time scale than when binding is irreversible. When the polymers do not overlap, allowing reversible binding increases the number of polymers adhered and hence the charge that the DNA can accommodate; in contrast, when overlaps occur, reversible binding reduces the amount of charge neutralized by the polymers.

DNA charge neutralization by linear polymers: Irreversible binding

We develop a deterministic mathematical model to describe the way in which polymers bind to DNA by considering the dynamics of the gap distribution that forms when polymers bind to a DNA plasmid. In so doing, we generalize existing theory to account for overlaps and binding cooperativity whereby the polymer binding rate depends on the size of the overlap. The proposed mean-field models are then solved using a combination of numerical and asymptotic methods. We find that overlaps lead to higher coverage and hence higher charge neutralizations, results which are more in line with recent experimental observations. Our work has applications to gene therapy where polymers are used to neutralize the negative charges of the DNA phosphate backbone, allowing condensation prior to delivery into the nucleus of an abnormal cell.

Molecular Design of Polyamines and Related Compounds for Controlled DNA Folding Transition

A library of designed DNA compaction agents based on multicationic polyamines, which can fold a DNA polymer chain into condensates with high density due to negative charge neutralization of DNA, are synthesized, and the structure-activity relationship in DNA compaction is investigated. It becomes obvious that the activity is strongly affected by non-electrostatic interactions with DNA, such as hydrophobic (hydrophilic), intercalation, and chirality effects, as well as by efficiency of charge interactions and geometry matching between charged groups of DNA and compaction agents.

The fractal structure of polycation–DNA complexes

We used static light scattering to obtain new measurements on the internal structure of aggregated non-viral gene-delivery particles in colloidal suspension. The vector particles are prepared by charge neutralization of plasmid DNA either by poly-L-lysine or by a Lipofectin/integrin-targeting peptide. We use established theories of the stability of colloidal particles and fractal concepts to explain the aggregation processes and demonstrate the existence of a new property (fractal dimension) of the aggregated vector particles. Aggregation is shown to produce particles with fractal dimensions in the range between 1.8 and 2.4; the former suggests a loose three-dimensional structure and the latter characterizes an aggregation process that leads to the formation of particles with tightly packed structures. We show that the fractal dimension of the vector particles is sensitive to changes in physicochemical conditions (ionic strength) of the buffer solution and propose that fractal dimension may provide a useful means of monitoring the physical state of non-viral delivery-vector particles during preparation and storage.