Heteromultivalency enables enhanced detection of nucleic acid mutations.

Detecting genetic mutations such as single nucleotide polymorphisms (SNPs) is necessary to prescribe effective cancer therapies, perform genetic analyses and distinguish similar viral strains. Traditionally, SNP sensing uses short oligonucleotide probes that differentially bind the SNP and wild-type targets. However, DNA hybridization-based techniques require precise tuning of the probe's binding affinity to manage the inherent trade-off between specificity and sensitivity. As conventional hybridization offers limited control over binding affinity, here we generate heteromultivalent DNA-functionalized particles and demonstrate optimized hybridization specificity for targets containing one or two mutations. By investigating the role of oligo lengths, spacer lengths and binding orientation, we reveal that heteromultivalent hybridization enables fine-tuned specificity for a single SNP and dramatic enhancements in specificity for two non-proximal SNPs empowered by highly cooperative binding. Capitalizing on these abilities, we demonstrate straightforward discrimination between heterozygous cis and trans mutations and between different strains of the SARS-CoV-2 virus. Our findings indicate that heteromultivalent hybridization offers substantial improvements over conventional monovalent hybridization-based methods.

Subtle changes in pH affect the packing and robustness of fatty acid bilayers.

Connecting molecular interactions to emergent properties is a goal of physical chemistry, self-assembly, and soft matter science. We show that for fatty acid bilayers, vesicle rupture tension, and permeability to water and ions are coupled to pH via alterations to lipid packing. A change in pH of one, for example, can halve the rupture tension of oleic acid membranes, an effect that is comparable to increasing lipid unsaturation in phospholipid systems. We use both experiments and molecular dynamics simulations to reveal that a subtle increase in pH can lead to increased water penetration, ion permeability, pore formation rates, and membrane disorder. For changes in membrane water content, oleic acid membranes appear to be more than a million times more sensitive to protons than to sodium ions. The work has implications for systems in which fatty acids are likely to be found, for example in the primitive cells on early Earth, biological membranes especially during digestion, and other biomaterials.

Free energy of micellization of dodecyl phosphocholine (DPC) from molecular simulation: Hybrid PEACH-BAR method.

A new method to extract the free energy of aggregation versus aggregate size from molecular simulation data is proposed and applied to a united atom model of the zwitterionic surfactant dodecyl phosphocholine in water. This system's slow dissociation rate and low critical micelle concentration (CMC of approximately 1-2 mM) make extraction of cluster free energies directly from simulation results using the "partition-enabled analysis of cluster histogram" (PEACH) method impractical. The new approach applies PEACH to a model with weakened attractions between aggregants, which allows sampling of a continuous range of cluster sizes, then recovers the free energy of aggregation under the original fully-attractive force field using the BAR free energy difference method. PEACH-BAR results are compared with free energy differences calculated via umbrella sampling, and are used to make predictions of CMC, average cluster size, and SAXS scattering profiles that are in fair agreement with experiment.

Bulk Self-Assembly of Giant, Unilamellar Vesicles.

The desire to create cell-like models for fundamental science and applications has spurred extensive effort toward creating giant unilamellar vesicles (GUVs). However, a route to selectively self-assemble GUVs in bulk has remained elusive. In bulk solution, membrane-forming molecules such as phospholipids, single-tailed surfactants, and block copolymers typically self-assemble into multilamellar, onion-like structures. So although self-assembly processes can form nanoscale unilamellar vesicles, scaffolding by droplets or surfaces is required to create GUVs. Here we show that it is possible to bulk self-assemble cell-sized GUVs with almost complete selectivity over other vesicle topologies. The seemingly paradoxical pair of features that enables this appears to be having very dynamic molecules at the nanoscale that create unusually rigid membranes. The resultant self-assembly pathway enables encapsulation of molecules and colloids and can also generate model primitive cells that can grow and divide.

Engineering DNA-Functionalized Nanostructures to Bind Nucleic Acid Targets Heteromultivalently with Enhanced Avidity.

Improving the affinity of nucleic acids to their complements is an important goal for many fields spanning from genomics to antisense therapy and diagnostics. One potential approach to achieving this goal is to use multivalent binding, which often boosts the affinity between ligands and receptors, as exemplified by virus-cell binding and antibody-antigen interactions. Herein, we investigate the binding of heteromultivalent DNA-nanoparticle conjugates, where multiple unique oligonucleotides displayed on a nanoparticle form a multivalent complex with a long DNA target containing the complementary sequences. By developing a strategy to spatially pattern oligonucleotides on a nanoparticle, we demonstrate that the molecular organization of heteromultivalent nanostructures is critical for effective binding; patterned particles have a ∼23 order-of-magnitude improvement in affinity compared to chemically identical particles patterned incorrectly. We envision that nanostructures presenting spatially patterned heteromultivalent DNA will offer important biomedical applications given the utility of DNA-functionalized nanostructures in diagnostics and therapeutics.

Competing factors in grain boundary loop shrinkage: Two-dimensional hard sphere colloidal crystals.

A grain boundary (GB) loop in a two-dimensional solid is the boundary of a domain or grain whose lattice orientation is mismatched with its uniform surroundings. Understanding the factors that influence the rate at which the interior of a GB loop relaxes to the orientation of its surroundings is an important step toward control and predictability of grain coarsening in general. Recent computational and experimental studies looking at the rate of GB loop shrinkage in two-dimensional colloidal hard sphere solids have uncovered contradictory trends: in experiments, GB loops with low misorientation angles shrank the fastest, while in simulations, they persisted the longest. In this study, the computational system's behavior is brought into qualitative agreement with the experimental results through increasing the lateral packing pressure, decreasing the domain size, and mimicking the experimental protocol used to form the GB loop. Small GB loops with the same misorientation, but displaying either a hexagonal or starlike grain shape depending on the orientation of their six dislocations, are shown to differ in their rates of shrinkage by two orders of magnitude. The evidence suggests that both the barrier to generating new dislocations as well as the pattern of dislocations formed by different GB loop preparation methods will determine which trend is observed.

Ordering of colloidal hard spheres under gravity: from monolayer to multilayer.

The phase behaviour of hard spheres confined by a gravitational potential to a thin layer (up to several monolayers) near a hard, flat surface is investigated using grand canonical Monte Carlo simulation. Depending on the strength of the gravitational field, the bottom monolayer of spheres may adopt uniform hexagonal order before, during, or after the growth of the second layer of particles. The crossover from ordering with a sparsely populated overlayer to ordering with almost one-third of the system's particles forming a second layer is observed upon decreasing the dimensionless Péclet number Pe = mgσ/kBT from 18 to 16. The particular sensitivity of the nature of the transition to particle size in this range is interpreted in terms of competing influences on the base layer structure by particles in the overlayer: promotion of order through increased pressure, versus stabilization of defects through occupation of low-lying sites on top of them. Simulations of grain boundaries between 2-D ordered domains of different orientation are used to correlate the degree of overlayer coverage to its effects on grain boundary stiffness as an indicator of defect free energy. Finally, we examine the structure of the ordered phases at coexistence over a range of gravitational strengths and find that orientational ordering of the second monolayer occurs along with first-order transition of the base layer at Pe = 8 but not at Pe = 10.

Simulations of NaCl Aggregation from Solution: Solvent Determines Topography of Free Energy Landscape.

The partition-enabled analysis of cluster histograms (PEACH) method is used to calculate the free energy surface of NaCl aggregation using cluster statistics from MD simulations of small systems (40-90 ions plus solvent) in four solvents. In all cases (pure methanol, pure water, and two methanol/water mixtures) NaCl clusters show a transition from amorphous to rocksalt structure with increasing cluster size. The crossover sizes, and the apparent kinetic barrier to ordering, increase with increasing water content. Implications for the proposed two-step mechanism of NaCl crystal nucleation (in which the ordered structure emerges from a large disordered cluster), and how this mechanism might depend on solvent and on degree of supersaturation, are discussed. In pure water, nonideal crowding effects that promote clustering are identified from systematic concentration-dependent deviations between simulation results and the PEACH model fit. In contrast, the ability of PEACH to fit aggregation statistics in mixed solvents is consistent with negligible interactions between ions in different clusters. © 2018 Wiley Periodicals, Inc.

Partitioning of Size-Mismatched Impurities to Grain Boundaries in 2d Solid Hard-Sphere Monolayers.

Computational studies have been carried out to investigate the equilibrium partitioning of size-mismatched impurities between the bulk solid and grain boundary (GB) environments in 2d hard-sphere monolayers. The solvent repacking Monte Carlo method and a new variation were used to exchange varying numbers and types of particles under conditions of fixed particle fugacities, allowing efficient sampling of impurity particle distributions even within the bulk solid. Measurements of GB stiffness depression arising from the impurities were made via the capillary fluctuation method and found to agree with calculations based on the Gibbs adsorption isotherm, providing a test of the internal consistency of the results. The dependence of the excess concentration at the GB on factors, including impurity size (diameter ratios λ = 0.5-4 times the majority host particle diameter), impurity concentration, grain misorientation angle, and packing pressure, was studied. In general, the affinity of impurity particles for GB increased with the difference between their size and the host particles, and varied with grain misorientation angle with a dependence reflecting the excess free area at the GB. Impurities with λ = 4 were exceptions to both these trends, due to their ability to substitute efficiently for six-coordinate host particles within the bulk and for five-coordinate host particles at dislocations in the grain boundaries. Comparison with results from an experimental study of mixed colloidal monolayers raises questions about how kinetic effects during grain coarsening might produce less impurity segregation to the GB than equilibrium exchange.

Simulations of grain boundaries between ordered hard sphere monolayer domains: Orientation-dependent stiffness and its correlation with grain coarsening dynamics.

The properties of grain boundaries (GBs) between ordered 2-d domains of a hard-sphere monolayer have been investigated using grand canonical Monte Carlo simulations. The capillary fluctuation method was used to determine the GB stiffness over a range of pressures, misorientations, and inclinations. Stiffness was found to be sensitive to misorientation (mismatch in the orientation angle of neighboring grains) but not to depend on inclination (angle between the boundary and the grain orientation). Excess area per GB length was observed to follow the same trend as stiffness with respect to grain misorientation and GB inclination angles. Dynamical studies of the evolution of bicrystalline or multicrystalline monolayers with simple geometries show that the calculated angle-dependent stiffnesses correlate well with the rate at which the evolving grain structure decreases the lengths of various GBs, in agreement with recent experimental results on monolayers of colloidal microspheres.

Size-asymmetrical Lennard-Jones solid solutions: Interstitials and substitutions.

We present simulation studies of solid solutions formed upon compression of mixtures of Lennard-Jones particles with diameter ratios 2:1 and 3:1. Grand canonical Monte Carlo (GCMC) and Gibbs-Duhem integration were used to determine the compositions of coexisting solid and liquid phases at several pressures and fixed temperature. Concentrations of small particles dissolved in interstitial sites of the large-particle lattice, under liquid-solid coexistence conditions, were determined directly from GCMC simulations. Indirect methods were used to calculate levels of small particles dissolved substitutionally, either singly or in plural, with the average number of small solutes occupying a lattice site vacated by a large particle increasing with higher pressure. In the cases studied, the fraction of small solutes occupying these substitutional sites was found to be small (2% or lower, depending on the mixture and conditions), but to stay roughly constant with increasing pressure. Structural and dynamic characteristics of the solid solutions are described and compared with reported characteristics of the related interstitial solid solution formed by hard spheres.

Binding, folding and insertion of a ß-hairpin peptide at a lipid bilayer surface: Influence of electrostatics and lipid tail packing.

Antimicrobial peptides (AMPs) act as host defenses against microbial pathogens. Here we investigate the interactions of SVS-1 (KVKVKVKVdPlPTKVKVKVK), an engineered AMP and anti-cancer ß-hairpin peptide, with lipid bilayers using spectroscopic studies and atomistic molecular dynamics simulations. In agreement with literature reports, simulation and experiment show preferential binding of SVS-1 peptides to anionic over neutral bilayers. Fluorescence and circular dichroism studies of a Trp-substituted SVS-1 analogue indicate, however, that it will bind to a zwitterionic DPPC bilayer under high-curvature conditions and folds into a hairpin. In bilayers formed from a 1:1 mixture of DPPC and anionic DPPG lipids, curvature and lipid fluidity are also observed to promote deeper insertion of the fluorescent peptide. Simulations using the CHARMM C36m force field offer complementary insight into timescales and mechanisms of folding and insertion. SVS-1 simulated at an anionic mixed POPC/POPG bilayer folded into a hairpin over a microsecond, the final stage in folding coinciding with the establishment of contact between the peptide's valine sidechains and the lipid tails through a "flip and dip" mechanism. Partial, transient folding and superficial bilayer contact are seen in simulation of the peptide at a zwitterionic POPC bilayer. Only when external surface tension is applied does the peptide establish lasting contact with the POPC bilayer. Our findings reveal the influence of disruption to lipid headgroup packing (via curvature or surface tension) on the pathway of binding and insertion, highlighting the collaborative effort of electrostatic and hydrophobic interactions on interaction of SVS-1 with lipid bilayers.

Cluster Free Energies from Simple Simulations of Small Numbers of Aggregants: Nucleation of Liquid MTBE from Vapor and Aqueous Phases.

We introduce a global fitting analysis method to obtain free energies of association of noncovalent molecular clusters using equilibrated cluster size distributions from unbiased constant-temperature molecular dynamics (MD) simulations. Because the systems simulated are small enough that the law of mass action does not describe the aggregation statistics, the method relies on iteratively determining a set of cluster free energies that, using appropriately weighted sums over all possible partitions of N monomers into clusters, produces the best-fit size distribution. The quality of these fits can be used as an objective measure of self-consistency to optimize the cutoff distance that determines how clusters are defined. To showcase the method, we have simulated a united-atom model of methyl tert-butyl ether (MTBE) in the vapor phase and in explicit water solution over a range of system sizes (up to 95 MTBE in the vapor phase and 60 MTBE in the aqueous phase) and concentrations at 273 K. The resulting size-dependent cluster free energy functions follow a form derived from classical nucleation theory (CNT) quite well over the full range of cluster sizes, although deviations are more pronounced for small cluster sizes. The CNT fit to cluster free energies yielded surface tensions that were in both cases lower than those for the simulated planar interfaces. We use a simple model to derive a condition for minimizing non-ideal effects on cluster size distributions and show that the cutoff distance that yields the best global fit is consistent with this condition.

Line Tension Assists Membrane Permeation at the Transition Temperature in Mixed-Phase Lipid Bilayers.

The umbrella sampling method has been used to evaluate the free energy profile for a large permeant moving through a lipid bilayer, represented using a coarse-grained simulation model, at and below its gel-fluid transition temperature. At the lipid transition temperature, determined to be 302 K for the MARTINI 2.0 model of DPPC, the permeation barrier for passage through an enclosed fluid domain embedded in a patch of gel was significantly lower than that for passage through a fluid stripe domain. In contrast, permeation through a fluid domain in a stripe geometry produced a free energy profile nearly identical to that of a gel-free fluid bilayer. In both cases, insertion of the permeant into a fluid domain coexisting with the gel phase led to a shift in phase composition, as lipids transitioned from fluid to gel to accommodate the area occupied by the permeant. In the case of the enclosed fluid domain, this transition produced a decrease in the length of the fluid-gel interface as the approximately circular fluid domain shrank. The observed decrease in the apparent permeation barrier, combined with an approximation for the change in interfacial length, enabled estimation of the interfacial line tension to be between 10 and 13 pN for this model. The permeation barrier was shown to drop even further in simulations performed at temperatures below the transition temperature. The results suggest a mechanism to explain the experimentally observed anomalous peak in the temperature-dependent permeability of lipid bilayers near their transition temperatures. The contribution of this mechanism toward the permeability of a gel phase containing a thermal distribution of fluid-phase domains is estimated using a simple statistical thermodynamic model.

Coarse-grained molecular simulations of the melting kinetics of small unilamellar vesicles.

Simulations of small unilamellar lipid bilayer vesicles have been performed to model their response to an instantaneous rise in temperature, starting from an initial low-temperature structure, to temperatures near or above the main chain transition temperature. The MARTINI coarse-grained force-field was used to construct slabs of gel-phase DPPC bilayers, which were assembled into truncated icosahedral structures containing 13,165 or 31,021 lipids. Equilibration at 280 K produced structures with several (5-8) domains, characterized by facets of lipids packed in the gel phase connected by disordered ridges. Instantaneous heating to final temperatures ranging from 290 K to 310 K led to partial or total melting over 500 ns trajectories, accompanied by changes in vesicle shape and the sizes and arrangements of remaining gel-phase domains. At temperatures that produced partial melting, the gel-phase lipid content of the vesicles followed an exponential decay, similar in form and timescale to the sub-microsecond phase of melting kinetics observed in recent ultrafast IR temperature-jump experiments. The changing rate of melting appears to be the outcome of a number of competing contributions, but changes in curvature stress arising from the expansion of the bilayer area upon melting are a major factor. The simulations give a more detailed picture of the changes that occur in frozen vesicles following a temperature jump, which will be of use for the interpretation of temperature-jump experiments on vesicles.

Grand canonical Monte Carlo using solvent repacking: Application to phase behavior of hard disk mixtures.

A new "solvent repacking Monte Carlo" strategy for performing grand canonical ensemble simulations in condensed phases is introduced and applied to the study of hard-disk systems. The strategy is based on the configuration-bias approach, but uses an auxiliary biasing potential to improve the efficiency of packing multiple solvent particles in the cavity formed by removing one large solute. The method has been applied to study the coexistence of ordered and isotropic phases in three binary mixtures of hard disks with a small mole fraction (xL < 0.02) of the larger "solute" component. A chemical potential of 12.81 ± 0.01 kBT was found to correspond to the freezing transition of the pure hard disk "solvent." Simulations permitted the study of partitioning of large disks between ordered and isotropic phases, which showed a distinct non-monotonic dependence on size; the isotropic phase was enriched approximately 10-fold, 20-fold, and 5-fold over the coexisting ordered phases at diameter ratios d = 1.4, 2.5, and 3, respectively. Mixing of large and small disks within both phases near coexistence was strongly non-ideal in spite of the dilution. Structures of systems near coexistence were analyzed to determine correlations between large disks' positions within each phase, the orientational correlation length of small disks within the fluid phases, and the nature of translational order in the ordered phase. The analyses indicate that the ordered phase coexists with an isotropic phase resembling a nanoemulsion of ordered domains of small disks, with large disks enriched at the disordered domain interfaces.

Molecular Simulation of the DPPE Lipid Bilayer Gel Phase: Coupling between Molecular Packing Order and Tail Tilt Angle.

The structural properties and thermal stability of dipalmitoylphosphatidylethanolamine (DPPE) in the ordered gel phase have been studied by molecular dynamics simulation using two force fields: the Berger united-atom model and the CHARMM C36 atomistic model. As is widely known, structural features are sensitive to the initial preparation of the gel phase structure, as some degrees of freedom are slow to equilibrate on the simulation time scale of hundreds of nanoseconds. In particular, we find that the degree of alignment of the lipids' glycerol backbones, which join the two hydrocarbon tails of each molecule, strongly affects the tilt angle of the tails in the resulting structures. Disorder in the backbone correlates with lower tilt angles: bilayer configurations initiated with aligned backbones produced tilt angles near 21° and 29° for the Berger and C36 force fields, respectively, while structures initiated with randomized backbone orientations showed average tilt angles of 7° and 18°, in closer agreement with the untilted structure observed experimentally. The transition temperature for the Berger force field gel bilayer has been determined by monitoring changes in width of gel phase stripe domains as a function of temperature and is 12 ± 5 K lower than the experimental value.

Simulation study of the permeability of a model lipid membrane at the fluid-solid phase transition.

When a range of lipid bilayers are melted to the disordered fluid phase from the (much less permeable) ordered gel phase, their permeability to a variety of permeants shows a peak at the transition temperature and drops off with increasing temperature, rather than just rising as melting proceeds. To explore this anomalous behavior, a simulated coarse-grained lipid membrane model that exhibits a phase transition upon expansion or compression was studied to determine how the permeation rate of a simple particle depends on the phase composition in the two-phase region and on particle size. The permeation rate and each phase's area fraction and area density could be directly calculated, along with the probability that the permeant would cross in either phase or in interfacial regions. For large permeants and system sizes, conditions could be found where permeability increases upon compression of the bilayer. Permeation was negligible in the gel phase and, in contrast to the predictions of the "leaky interface" hypothesis, was not enriched in interfacial regions. The anomalous effect could instead be attributed to an increase in the area per lipid of fluid-phase domains. This result motivated a model for the decrease in effective permeability barrier through fluid-phase domains arising from a decrease in the length of the gel/fluid interface at the midpoint of a permeation event.

Quantum dots encapsulated within phospholipid membranes: phase-dependent structure, photostability, and site-selective functionalization.

Lipid vesicle encapsulation is an efficient approach to transfer quantum dots (QDs) into aqueous solutions, which is important for renewable energy applications and biological imaging. However, little is known about the molecular organization at the interface between a QD and lipid membrane. To address this issue, we investigated the properties of 3.0 nm CdSe QDs encapsulated within phospholipid membranes displaying a range of phase transition temperatures (Tm). Theoretical and experimental results indicate that the QD locally alters membrane structure, and in turn, the physical state (phase) of the membrane controls the optical and chemical properties of the QDs. Using photoluminescence, ICP-MS, optical microscopy, and ligand exchange studies, we found that the Tm of the membrane controls optical and chemical properties of lipid vesicle-embedded QDs. Importantly, QDs encapsulated within gel-phase membranes were ultrastable, providing the most photostable non-core/shell QDs in aqueous solution reported to date. Atomistic molecular dynamics simulations support these observations and indicate that membranes are locally disordered displaying greater disordered organization near the particle-solution interface. Using this asymmetry in membrane organization near the particle, we identify a new approach for site-selective modification of QDs by specifically functionalizing the QD surface facing the outer lipid leaflet to generate gold nanoparticle-QD assemblies programmed by Watson-Crick base-pairing.

Simulation studies of structure and edge tension of lipid bilayer edges: effects of tail structure and force-field.

Molecular dynamics simulations of lipid bilayer ribbons have been performed to investigate the structures and line tensions associated with free bilayer edges. Simulations carried out for dioleoyl phosphatidylcholine with three different force-field parameter sets yielded edge line tensions of 45 ± 2 pN, over 50% greater than the most recently reported experimentally determined value for this lipid. Edge tensions obtained from simulations of a series of phosphatidylcholine lipid bilayer ribbons with saturated acyl tails of length 12-16 carbons and with monounsaturated acyl tails of length 14-18 carbons could be correlated with the excess area associated with forming the edge, through a two-parameter fit. Saturated-tail lipids underwent local thickening near the edge, producing denser packing that correlated with lower line tensions, while unsaturated-tail lipids showed little or no local thickening. In a dipalmitoyl phosphatidylcholine ribbon initiated in a tilted gel-phase structure, lipid headgroups tended to tilt toward the nearer edge producing a herringbone pattern, an accommodation that may account for the reported edge-induced stabilization of an ordered structure at temperatures near a lipid gel-fluid phase transition.