Effects of Structural Isomers of Spermine on the Higher-Order Structure of DNA and Gene Expression.

Polyamines are involved in various biological functions, including cell proliferation, differentiation, gene regulation, etc. Recently, it was found that polyamines exhibit biphasic effects on gene expression: promotion and inhibition at low and high concentrations, respectively. Here, we compared the effects of three naturally occurring tetravalent polyamines, spermine (SPM), thermospermine (TSPM), and N4-aminopropylspermidine (BSPD). Based on the single DNA observation with fluorescence microscopy together with measurements by atomic force microscopy revealed that these polyamines induce shrinkage and then compaction of DNA molecules, at low and high concentrations, respectively. We also performed the observation to evaluate the effects of these polyamine isomers on the activity of gene expression by adapting a cell-free luciferase assay. Interestingly, the potency of their effects on the DNA conformation and also on the inhibition of gene expression activity indicates the highest for TSPM among spermine isomers. A numerical evaluation of the strength of the interaction of these polyamines with negatively charged double-strand DNA revealed that this ordering of the potency corresponds to the order of the strength of the attractive interaction between phosphate groups of DNA and positively charged amino groups of the polyamines.

Specific effects of antitumor active norspermidine on the structure and function of DNA.

We compared the effects of trivalent polyamines, spermidine (SPD) and norspermidine (NSPD), a chemical homologue of SPD, on the structure of DNA and gene expression. The chemical structures of SPD and NSPD are different only with the number of methylene groups between amine groups, [N-3-N-4-N] and [N-3-N-3-N], respectively. SPD plays vital roles in cell function and survival, including in mammals. On the other hand, NSPD has antitumor activity and is found in some species of plants, bacteria and algae, but not in humans. We found that both polyamines exhibit biphasic effect; enhancement and inhibition on in vitro gene expression, where SPD shows definitely higher potency in enhancement but NSPD causes stronger inhibition. Based on the results of AFM (atomic force microscopy) observations together with single DNA measurements with fluorescence microscopy, it becomes clear that SPD tends to align DNA orientation, whereas NSPD induces shrinkage with a greater potency. The measurement of binding equilibrium by NMR indicates that NSPD shows 4-5 times higher affinity to DNA than SPD. Our theoretical study with Monte Carlo simulation provides the insights into the underlying mechanism of the specific effect of NSPD on DNA.

Cooperative standing-horizontal-standing reentrant transition for numerous solid particles under external vibration.

We report the collective behavior of numerous plastic bolt-like particles exhibiting one of two distinct states, either standing stationary or horizontal accompanied by tumbling motion, when placed on a horizontal plate undergoing sinusoidal vertical vibration. Experimentally, we prepared an initial state in which all of the particles were standing except for a single particle that was placed at the center of the plate. Under continuous vertical vibration, the initially horizontal particle triggers neighboring particles to fall over into a horizontal state through tumbling-induced collision, and this effect gradually spreads to all of the particles, i.e., the number of horizontal particles is increased. Interestingly, within a certain range of vibration intensity, almost all of the horizontal particles revert back to standing in association with the formation of apparent 2D hexagonal dense-packing. Thus, phase segregation between high and low densities, or crystalline and disperse domains, of standing particles is generated as a result of the reentrant transition. The essential features of such cooperative dynamics through the reentrant transition are elucidated with a simple kinetic model. We also demonstrate that an excitable wave with the reentrant transition is observed when particles are situated in a quasi-one-dimensional confinement on a vibrating plate.

Localization switching of a large object in a crowded cavity: A rigid/soft object prefers surface/inner positioning.

For living cells in the real world, a large organelle is commonly positioned in the inner region away from membranes, such as the nucleus of eukaryotic cells, the nucleolus of nuclei, mitochondria, chloroplast, Golgi body, etc. It contradicts the expectation by the current depletion-force theory in that the larger particle should be excluded from the inner cell space onto cell boundaries in a crowding media. Here we simply model a sizable organelle as a soft-boundary large particle allowing crowders, which are smaller hard spheres in the model, to intrude across its boundary. The results of Monte Carlo simulation indicate that the preferential location of the larger particle switches from the periphery into the inner region of the cavity by increasing its softness. An integral equation theory is further developed to account for the structural features of the model, and the theoretical predictions are found consistent with our simulation results.

A toy model for nucleus-sized crowding confinement.

We conduct Monte Carlo simulations to understand the spatial distribution of a polymer molecule confined within a rigid spherical capsule under crowding conditions, via a bead-spring chain model. To adjust the crowding level, the polymer is mixed with spherical crowders. As the interior of the capsule becomes more crowded, chain monomers tend to move to the capsule boundary under the penalty of conformational entropy. By incorporating some attraction between monomers and crowders, the polymer chain moves away from the capsule boundary. The interplay, between the conformational entropy, DNA-protein interaction, and molecular crowding induced depletion force between the chain and capsule boundary, may be essential to elucidate the heterogeneous chromatin structure in nuclei. Furthermore, the effects of chain length and size disparity between the monomers and the crowders are also investigated preliminarily.

Phase behavior under a noncentrosymmetric interaction: shifted-charge colloids investigated by Monte Carlo simulation.

Using Monte Carlo simulations, we investigate the structural characteristics of an interacting hard-sphere system with shifted charge to elucidate the effect of the noncentrosymmetric interaction on its phase behavior. Two different phase transitions are identified for this model system. With increasing volume fraction, an abrupt liquid-to-crystal transition first occurs at a significantly lower volume fraction than in centrosymmetrically charged systems. This is due to the stronger effective interparticle repulsion caused by the additional charge anisotropy. Moreover, within the crystal state at higher volume fraction, the system further undergoes a continuous disorder-to-order transition with respect to charge orientation. Detailed analyses in this work disclose the nature of these transitions, and orientation fluctuation can cause noncentrosymmetric unit cells. The dependence of crystal formation and orientational ordering on temperature was also examined. These findings indicate that the noncentrosymmetric interaction in this work results in additional freedoms to fine-tune the phase diagram and increase the functionalities of materials. Moreover, these model studies are essential to advance the future understanding regarding the fundamental physiochemical properties of novel Janus colloidal particles and protein crystallization conditions.

Rigidity of a spherical capsule switches the localization of encapsulated particles between inner and peripheral regions under crowding condition: simple model on cellular architecture.

We have investigated the inhomogeneous interior of confined spherical cavities as capsules containing encapsulated binary hard sphere mixtures for different compositions and cavity wall rigidity. Such a greatly simplified model manifests the effects of macromolecular crowding arising from excluded volume interactions in a tiny cell or a cellular nucleus. By fixing the number of large particles, the level of crowding is adjusted by changing the amount of small hard spheres in the cavity. For a rigid cavity, large spheres tend to pack in liquid-like order apart from the surface to the center of the cavity as the crowding level is increased. Whereas, for a soft cavity, larger spheres tend to blend with small spheres in the peripheral region at near the boundary of the cavity, and are susceptible to be depleted from the interior of the cavity as the cavity becomes more crowded. These results may help future elucidation of the thermodynamic pathways to stabilize the inhomogeneous structure of mixtures confined in cavities, such as the derepression of genome materials around the interior rim of the nucleus in a cancerous cell.

Conformational effect on small angle neutron scattering behavior of interacting polyelectrolyte solutions: a perspective of integral equation theory.

We present small angle neutron scattering (SANS) measurements of deuterium oxide (D(2)O) solutions of linear and star sodium poly(styrene sulfonate) (NaPSS) as a function of polyelectrolyte concentration. Emphasis is on understanding the dependence of their SANS coherent scattering cross section I(Q) on the molecular architecture of single polyelectrolyte. The key finding is that for a given concentration, star polyelectrolytes exhibit more pronounced characteristic peaks in I(Q), and the position of the first peak occurs at a smaller Q compared to their linear counterparts. Based on a model of integral equation theory, we first compare the SANS experimental I(Q) of salt-free polyelectrolyte solutions with that predicted theoretically. Having seen their satisfactory qualitative agreement, the dependence of counterion association behavior on polyelectrolyte geometry and concentration is further explored. Our predictions reveal that the ionic environment of polyelectrolyte exhibits a strong dependence on polyelectrolyte geometry at lower polyelectrolyte concentration. However, when both linear and star polyelectrolytes exceed their overlap concentrations, the spatial distribution of counterion is found to be essentially insensitive to polyelectrolyte geometry due to the steric effect.

Elucidation of spin echo small angle neutron scattering correlation functions through model studies.

Several single-modal Debye correlation functions to approximate part of the overall Debey correlation function of liquids are closely examined for elucidating their behavior in the corresponding spin echo small angle neutron scattering (SESANS) correlation functions. We find that the maximum length scale of a Debye correlation function is identical to that of its SESANS correlation function. For discrete Debye correlation functions, the peak of SESANS correlation function emerges at their first discrete point, whereas for continuous Debye correlation functions with greater width, the peak position shifts to a greater value. In both cases, the intensity and shape of the peak of the SESANS correlation function are determined by the width of the Debye correlation functions. Furthermore, we mimic the intramolecular and intermolecular Debye correlation functions of liquids composed of interacting particles based on a simple model to elucidate their competition in the SESANS correlation function. Our calculations show that the first local minimum of a SESANS correlation function can be negative and positive. By adjusting the spatial distribution of the intermolecular Debye function in the model, the calculated SESANS spectra exhibit the profile consistent with that of hard-sphere and sticky-hard-sphere liquids predicted by more sophisticated liquid state theory and computer simulation.

Contrast variation in spin-echo small angle neutron scattering.

The use of contrast variation in spin-echo small angle neutron scattering (SESANS) experiments is discussed for the case of colloidal structural investigation. On the basis of calculations for several model systems, we find that the contrast variation SESANS technique, in terms of the measured SESANS correlation function G(z), is not sensitive to the structural characteristics of colloidal suspensions consisting of particles with uniform scattering length density profiles. However, its ability to resolve structural heterogeneity, at both intra-colloidal and inter-colloidal length scales, is clearly demonstrated. The prospect of using this new technique to investigate structural information that is difficult to probe in other ways is also explored.

Spatial distribution of intra-molecular water and polymeric components in polyelectrolyte dendrimers revealed by small angle scattering investigations.

An experimental scheme using contrast variation small angle neutron scattering technique is developed to investigate the structural characteristics of amine-terminated poly(amidoamine) dendrimers solutions. Using this methodology, we present the dependence of both the intra-dendrimer water and the polymer distribution on molecular protonation, which can be precisely adjusted by tuning the pH of the solution. Assuming spherical symmetry of the spatial arrangement of the constituent components of dendrimer, and that the atomic ratio of hydrogen-to-deuterium for the solvent residing within the cavities of dendrimer is identical to that for the solvent outside the dendrimer, the intra-dendrimer water distribution along the radial direction is determined. Our result clearly reveals an outward relocation of the peripheral groups, as well as enhanced intra-dendrimer hydration, upon increasing the molecular protonation and, therefore, allows the determination of segmental backfolding in a quantitative manner. The connection between these charge-induced structural changes and our recently observed progressively active segmental dynamics is also discussed.

Prospect for characterizing interacting soft colloidal structures using spin-echo small angle neutron scattering.

Spin-echo small angle neutron scattering (SESANS) provides a new experimental tool for structural investigation. Due to the action of spin-echo encoding, SESANS measures a spatial correlation function in real space, as opposed to the structure factor S(Q), I(Q), in momentum (Q) space measured by conventional small angle neutron scattering. To establish the usefulness of SESANS in structural characterization, particularly for interacting colloidal suspensions, we have previously conducted a theoretical study of the SESANS correlation functions for model systems consisting of particles with uniform density profiles [X. Li, C.-Y. Shew, Y. Liu, R. Pynn, E. Liu, K. W. Herwig, G. S. Smith, J. L. Robertson, and W.-R. Chen J. Chem. Phys. 132, 174509 (2010)]. Within the same framework, we explore in the present paper the prospect of using SESANS to investigate the structural characteristics of colloidal systems consisting of particles with nonuniform intraparticle mass distribution. As an example, a Gaussian model of interacting soft colloids is used to investigate the manifestation of structural softness in a SESANS measurement. The exploration shows a characteristically different SESANS correlation function for interacting soft colloids, in comparison to that of a uniform hard sphere system. The difference arises from the Abel transform imbedded in the mathematical formalism bridging the SESANS spectra and the spatial autocorrelation function.

Water distributions in polystyrene-block-poly[styrene-g-poly(ethylene oxide)] block grafted copolymer system in aqueous solutions revealed by contrast variation small angle neutron scattering study.

We develop an experimental approach to analyze the water distribution around a core-shell micelle formed by polystyrene-block-poly[styrene-g-poly(ethylene oxide (PEO)] block copolymers in aqueous media at a fixed polymeric concentration of 10 mg/ml through contrast variation small angle neutron scattering (SANS) study. Through varying the D(2)O/H(2)O ratio, the scattering contributions from the water molecules and the micellar constituent components can be determined. Based on the commonly used core-shell model, a theoretical coherent scattering cross section incorporating the effect of water penetration is developed and used to analyze the SANS I(Q). We have successfully quantified the intramicellar water distribution and found that the overall micellar hydration level increases with the increase in the molecular weight of hydrophilic PEO side chains. Our work presents a practical experimental means for evaluating the intramacromolecular solvent distributions of general soft matter systems.

Theoretical studies on the structure of interacting colloidal suspensions by spin-echo small angle neutron scattering.

The application of the spin-echo small angle neutron scattering (SESANS) technique for structural characterization of interacting colloidal suspensions is considered in this work. The framework to calculate the theoretical SESANS correlation function is briefly laid out. A general discussion regarding the features of the SESANS correlation functions obtained from different model systems is presented. In comparison with conventional elastic scattering tools operating at the same length scale, our mean-field calculations, based on a monodisperse spherical colloidal system, show that the real-space measurement provided by SESANS presents a powerful probe for studying the intercolloid potential. The reason of this sensitivity is discussed from the standpoint of way, in which how the spatial correlations are manifested in different neutron scattering implementations. This study leads to a better understanding regarding the distinction between SANS and SESANS.

Loosening of DNA/polycation complexes by synthetic polyampholyte to improve the transcription efficiency: effect of charge balance in the polyampholyte.

High mobililty group proteins are amphoteric nuclear proteins that are known to unfold chromatin to stimulate transcription. To mimic their structures, we synthesized the novel polyethylene glycol (PEG) derivatives, PEG-ACs, consisting of both amino- and carboxyl-pendants in various ratios, and their loosening and transcription-improving activity on the DNA complex was examined. Fluorescence anisotropy measurement revealed that anionic PEG-ACs with more carboxyls than amines could efficiently loosen the DNA/polyethyleneimine complex. Those anionic PEG-ACs showing a loosening effect on the DNA complex evidently increased the transcription rate to >20 times higher than that of the original complex, probably owing to the facilitated approach of transcriptional factors to the DNA segments in the loosened complexes. The complexes with anionic PEG-ACs also showed improved transgene expression level on the cultured cells, indicating the effectiveness of improving transcriptional activity to attain a high extragene expression by the plasmid complex. The loosening mechanism of DNA/polycation complexes was investigated with a simplified model via Monte Carlo simulation to discern the difference in the presence of cationic polyampholytes, anionic polyampholytes, and polyanions.

Effect of counterion valence on the pH responsiveness of polyamidoamine dendrimer structure.

An accurate determination of the structure characteristics of protonated generation 5 polyamidoamine dendrimers in aqueous solution has been conducted by analyzing the small angle neutron scattering databased on a statistical mechanics model. In our investigation, the primary focus is to elucidate the effect of counterion valence on the counterion association and its impact on the intramolecular density profile within a dendrimer. In the range of our study for molecular protonation, a strong dependence of the structural properties of charged dendrimers on counterion valence is revealed. Our findings indicate that the association of a large amount of divalent counterions significantly reduces the effective charge of a dendrimer molecule. Surprisingly, no discernible transition of the density distribution profile is observed for the dendrimer charged by D(2)SO(4), as opposed to our previous observation of a pronounced transition in intramolecular density profile for the dendrimer charged by DCl. These findings may be understood from the thermodynamic processes of counterions.

A Monte Carlo algorithm for computing spin echo small angle neutron scattering correlation functions in real space: Hard sphere liquids.

A Monte Carlo algorithm is developed to compute the autocorrelation function of liquids and the corresponding spatial correlation function from spin echo small angle neutron scattering (SESANS) spectra. The accuracy of the simulation algorithm is tested with isolated hard spheres and single dumbbells consisting of two hard spheres separated by a given distance. The simulation results accurately reproduce the exact expressions of these two models. To further test the algorithm for many-body systems, two liquid models are considered including hard sphere fluids and hard spheres with an attractive tail. The many-particle Monte Carlo simulation is carried out to obtain the ensemble average of these correlation functions. Meanwhile, the Percus-Yevic (PY) integral equation theory is resorted to compute the autocorrelation function and SESANS spatial correlation function for a density that the PY theory is reasonably applicable. The agreement between simulation and theory indicates that the algorithm is quite robust and can be extended to more complex fluids in the future. Furthermore, we find that the SESANS spatial correlation function is highly sensitive to the interaction potential between particles, which may serve as a useful tool to explore particle interactions in a liquid.

Investigation of morphology of an enneadic particle system confined onto a spherical surface through Monte Carlo simulation.

The morphology of an enneadic particle system confined to a spherical surface is investigated through Monte Carlo simulation. The model assumes nine hard spheres adsorbed onto a spherical surface of the same diameter. In addition to hard core repulsion, these nine particles interact through Coulomb repulsion or Lennard-Jones potential. At a low enough temperature, the ordered structure of the enneadic particle system exhibits a symmetric arrangement corresponding to the D(3h) point group and is similar to hexagonal close packing. The nine particles can be divided into three groups, and each group occupies an equilateral triangle along with the same principle axis. The top and bottom triangles form the mirror image and the middle triangle of a different size rotates 60 degrees from the other two triangles. This symmetry is independent of interaction potentials but the two interaction potentials deform the ordered structure in a slightly different way. From the analysis of monomers of inertia, we notice that Coulombic particles compress the ordered morphology to an oblate shape, whereas Lennard-Jones particles display an elongated prolate morphology. By use of the analysis of a simple model, we find that these fine structures tend to lower the total energy of the system. Moreover, the calculation based on the temperature dependent heat capacity of many noninteracting enneadic clusters suggests that large interaction strengths are essential to sustain the highly symmetric ordered structure.

Improvement of transcription- and transfection-efficiency by synthetic polyampholytes.

Water-soluble PEG derivatives having both amino- and carboxyl-pendants (PEG-ACs) were synthesized, and examined for their transcription- and transfection-enhancing activity on DNA/polycation complexes. PEG-AC could be deposited onto the surface of DNA/polyethylenimine(PEI) complexes, and enhanced their transcriptional activity. Fluorescence anisotropy study showed that the amphoteric PEG-AC loosened the tightly compacted DNA/PEI complex to facilitate the approach of transcriptional factors. Transcriptional activity of the complex was in good correlation with its transgene expression efficiency, indicating the importance of improvement in transcription for effective gene transfection.