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Liquid-liquid phase separation (LLPS) in macromolecular solutions (e.g., coacervation) is relevant both to technology and to the process of mesoscale structure formation in cells. The LLPS process is characterized by a phase diagram, i.e., binodal lines in the temperature/concentration plane, which must be quantified to predict the system's behavior. Experimentally, this can be difficult due to complications in handling the dense macromolecular phase. Here, we develop a method for accurately quantifying the phase diagram without direct handling: We confine the sample within micron-scale, water-in-oil emulsion droplets and then use precision fluorescent imaging to measure the volume fraction of the condensate within the droplet. We find that this volume fraction grows linearly with macromolecule concentration; thus, by applying the lever rule, we can directly extract the dense and dilute binodal concentrations. We use this approach to study a model LLPS system of self-assembled, fixed-valence DNA particles termed nanostars (NSs). We find that temperature/concentration phase diagrams of NSs display, with certain exceptions, a larger co-existence regime upon increasing salt or valence, in line with expectations. Aspects of the measured phase behavior validate recent predictions that account for the role of valence in modulating the connectivity of the condensed phase. Generally, our results on NS phase diagrams give fundamental insight into limited-valence phase separation, while the method we have developed will likely be useful in the study of other LLPS systems.

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The reflectin proteins have been extensively studied for their role in reflectance in cephalopods. In the recently evolved Loliginid squids, these proteins and the structural color they regulate are dynamically tunable, enhancing their effectiveness for camouflage and communication. In these species, the reflectins are found in highest concentrations within the structurally tunable, membrane enclosed, periodically stacked lamellae of subcellular Bragg reflectors and in the intracellular vesicles of specialized skin cells known as iridocytes and leuocophores, respectively. To better understand the interactions between the reflectins and the membrane structures that encompass them, we analyzed the interactions of two purified reflectins with synthetic phospholipid membrane vesicles similar in composition to cellular membranes, using confocal fluorescence microscopy and dynamic light scattering. The purified recombinant reflectins were found to drive multivalent vesicle agglomeration in a ratio-dependent and saturable manner. Extensive proteolytic digestion terminated with PMSF of the reflectin A1-vesicle complexes triggered energetic membrane rearrangement, resulting in vesicle fusion, fission, and tubulation. This behavior contrasted markedly with that of vesicles complexed with reflectin C, from which PMSF-terminated proteolysis only released the original size vesicles. Clues to the basis for this difference, residing in significant differences between the structures of the two reflectins, led to the suggestion that specific reflectin-membrane interactions may play a role in the ontogenetic formation, long-term maintenance, and/or dynamic behavior of their biophotonically active host membrane nanostructures. Similar energetic remodeling has been associated with osmotic stress in other membrane systems, suggesting a path to reconstitution of the biophotonic system in vitro.

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The classic picture of soft material mechanics is that of rubber elasticity, in which material modulus is related to the entropic elasticity of flexible polymeric linkers. The rubber model, however, largely ignores the role of valence (i.e., the number of network chains emanating from a junction). Recent work predicts that valence, and particularly the Maxwell isostatic point, plays a key role in determining the mechanics of semiflexible polymer networks. Here, we report a series of experiments confirming the prominent role of valence in determining the mechanics of a model system. The system is based on DNA nanostars (DNAns): multiarmed, self-assembled nanostructures that form thermoreversible equilibrium gels through base pair-controlled cross-linking. We measure the linear and nonlinear elastic properties of these gels as a function of DNAns arm number, f, and concentration [DNAns]. We find that, as f increases from three to six, the gel's high-frequency plateau modulus strongly increases, and its dependence on [DNAns] transitions from nonlinear to linear. Additionally, higher-valence gels exhibit less strain hardening, indicating that they have less configurational freedom. Minimal strain hardening and linear dependence of shear modulus on concentration at high f are consistent with predictions for isostatic systems. Evident strain hardening and nonlinear concentration dependence of shear modulus suggest that the low-f networks are subisostatic and have a transient, potentially fractal percolated structure. Overall, our observations indicate that network elasticity is sensitive both to entropic elasticity of network chains and to junction valence, with an apparent isostatic point [Formula: see text] in agreement with the Maxwell prediction.

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Liquid-liquid phase separation of a polymer-rich phase from a polymer-dilute solution, known generally as coacervation, has been observed in a variety of biomolecular systems. Understanding of this process, and the properties of the resulting liquid, has been hampered in typical systems by the complexity of the components and of the intermolecular interactions. Here, we examine a single-component system comprised entirely of DNA, in which tetravalent DNA nanostar particles condense into liquids through attractive bonds formed from basepairing interactions. We measure the density, viscosity, particle self-diffusion, and surface tension of NS-liquid droplets. The sequence- and salt-dependent thermodynamics of basepairing accounts for most properties, particularly indicating that particle transport is an activated process whose barrier is the breaking of a single bond, and that very few bonds are broken at the surface. However, more complex effects are also seen. The relation of density to salt shows that electrostatic screening compacts the NS particles. Further, the interrelation of the transport properties indicates a breakdown of the Stokes-Einstein relation. This observation, in concert with the low surface tension and single-bond transport barrier, suggests this DNA liquid has a heterogeneous, clustered structure that is likely enabled by internal NS particle flexibility. We discuss these results in comparison to other coacervate systems.

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Neurotransmitters are small molecules that orchestrate complex patterns of brain activity. Unfortunately, there exist few sensors capable of directly detecting individual neurotransmitters. Those sensors that do exist are either unspecific or fail to capture the temporal or spatial dynamics of neurotransmitter release. DNA-stabilized silver nanoclusters (DNA-AgNCs) are a new class of biocompatible, fluorescent nanostructures that have recently been shown to offer promise as biosensors. In this work, we identify two different DNA sequences that form dopamine-sensitive nanoclusters. We demonstrate that each sequence supports two distinct DNA-AgNCs capable of providing specific, ratiometric fluorescent sensing of dopamine concentration in vitro. DNA-Ag nanoclusters therefore offer a novel, low-cost approach to quantification of dopamine, creating the potential for real-time monitoring in vivo.

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DNA-stabilized silver nanoclusters (AgNCs), the fluorescence emission of which can rival that of typical organic fluorophores, have made possible a new class of label-free molecular beacons for the detection of single-stranded DNA. Like fluorophore-quencher molecular beacons (FQ-MBs) AgNC-based molecular beacons (AgNC-MBs) are based on a single-stranded DNA that undergoes a conformational change upon binding a target sequence. The new conformation exposes a stretch of single-stranded DNA capable of hosting a fluorescent AgNC upon reduction in the presence of Ag(+) ions. The utility of AgNC-MBs has been limited, however, because changing the target binding sequence unpredictably alters cluster fluorescence. Here we show that the original AgNC-MB design depends on bases in the target-binding (loop) domain to stabilize its AgNC. We then rationally alter the design to overcome this limitation. By separating and lengthening the AgNC-stabilizing domain, we create an AgNC-hairpin probe with consistent performance for arbitrary target sequence. This new design supports ratiometric fluorescence measurements of DNA target concentration, thereby providing a more sensitive, responsive and stable signal compared to turn-on AgNC probes. Using the new design, we demonstrate AgNC-MBs with nanomolar sensitivity and singe-nucleotide specificity, expanding the breadth of applicability of these cost-effective probes for biomolecular detection.

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Rod-like particles form solutions of technological and biological importance. In particular, biofilaments such as actin and microtubules are known to form a variety of phases, both in vivo and in vitro, whose appearance can be controlled by depletion, confinement, and electrostatic interactions. Here, we utilize DNA nanotubes to undertake a comprehensive study of the effects of those interactions on two particular rod-like phases: a 2D nematic phase consisting of aligned rods pressed against a glass surface, and a 3D bundled network phase. We experimentally measure the stability of these two phases over a range of depletant concentrations and ionic strengths, finding that the 2D phase is slightly more stable than the 3D phase. We formulate a quantitative model of phase stability based on consideration of pairwise rod-rod and rod-surface interactions; notably, we include a careful accounting of solution electrostatics interactions using an effective-charge strategy. The model is relatively simple and contains no free parameters, yet predicts phase boundaries in good agreement with the experiment. Our results indicate that electrostatic interactions, rather than depletion, are largely responsible for the enhanced stability of the 2D phase. This work provides insight into the polymorphism of rod-like solutions, indicating why certain phases appear, and providing a means (and a predictive model) for controlling those phases.

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It is well-known that even small perturbations of the DNA sequence can drastically and unpredictably disrupt or alter the fluorescence of DNA-stabilized silver nanoclusters (DNA-AgNCs). Understanding how the structure of DNA affects the nanocluster that it stabilizes is the key to rationalizing such effects. We approach this challenge by strategically modifying the stem sequence of a hairpin DNA that hosts a spectrally pure, red-emitting nanocluster. Most of our modifications (base composition, sequence orientation, and loop location) reduce AgNC fluorescence in purity and shift it in wavelength, but one modification (appending poly(thymidine) to the 3' end of the stem) is inert with respect to fluorescence. Microfluidic capillary electrophoresis reveals that all of the modifications induce conformational changes of the DNA and that the original, spectrally pure nanocluster exists in two structurally distinct conformations. Interestingly, appending five or more thymidines, despite having no effect on fluorescence, eliminates this structural degeneracy. To explain this result, we propose that the original spectrally pure cluster is stabilized by a pair of hairpins whose stems can arrange in either a cis or trans orientation. Finally, we quantify the extent to which thymidine appendages of different lengths can be used to fine-tune the electrophoretic mobility of DNA-AgNC.

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Heparan sulfate (HS) is a ubiquitous glycosaminoglycan that serves as a cellular attachment site for a number of significant human pathogens, including respiratory syncytial virus (RSV), human parainfluenza virus 3 (hPIV3), and herpes simplex virus (HSV). Decoy receptors can target pathogens by binding to the receptor pocket on viral attachment proteins, acting as 'molecular sinks' and preventing the pathogen from binding to susceptible host cells. Decoy receptors functionalized with HS could bind to pathogens and prevent infection, so we generated decoy liposomes displaying HS-octasaccharide (HS-octa). These decoy liposomes significantly inhibited RSV, hPIV3, and HSV infectivity in vitro to a greater degree than the original HS-octa building block. The degree of inhibition correlated with the density of HS-octa displayed on the liposome surface. Decoy liposomes with HS-octa inhibited infection of viruses to a greater extent than either full-length heparin or HS-octa alone. Decoy liposomes were effective when added prior to infection or following the initial infection of cells in vitro. By targeting the well-conserved receptor-binding sites of HS-binding viruses, decoy liposomes functionalized with HS-octa are a promising therapeutic antiviral agent and illustrate the utility of the liposome delivery platform.

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DNA-stabilized fluorescent silver nanoclusters (AgNC DNA) are a new class of fluorophore that are formed by sequence specific interactions between silver and single-stranded DNA. By incorporating both target-binding and fluorescent-reporting sequences into a single synthetic DNA oligomer, AgNC DNA probes eliminate the need to conjugate dye or quencher molecules. In this study, we modify a AgNC DNA probe to demonstrate single-color multiplexed detection of DNA targets. We show that appending different lengths of poly-dT to the probe sequences tunes the electrophoretic mobility of AgNC DNA probes without affecting their fluorescence spectra. We use this to introduce a set of AgNC DNA probes selective for Hepatitis A, B and C target sequences that can be processed together in a simple, single-step protocol and distinguished with a resolution of 3.47 and signal to noise ratio of 17.23 in under 10 seconds by microfluidic capillary electrophoresis.

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We measure the stiffness of tiled DNA nanotubes (HX-tubes) as a function of their (defined) circumference by analyzing their micrometer-scale thermal deformations using fluorescence microscopy. We derive a model that relates nanoscale features of HX-tube architecture to the measured persistence lengths. Given the known stiffness of double-stranded DNA, we use this model to constrain the average spacing between and effective stiffness of individual DNA duplexes in the tube. A key structural feature of tiled nanotubes that can affect stiffness is their potential to form with discrete amounts of twist of the DNA duplexes about the tube axis (supertwist). We visualize the supertwist of HX-tubes using electron microscopy of gold nanoparticles, attached to specific sites along the nanotube. This method reveals that HX-tubes tend not to form with supertwist unless forced by sequence design, and, even when forced, supertwist is reduced by elastic deformations of the underlying DNA lattice. We compare the hybridization energy gained upon closing a duplex sheet into a tube with the elastic energy paid for deforming the sheet to allow closure. In estimating the elastic energy we account for bending and twisting of the individual duplexes as well as shearing between them. We find the minimum supertwist state has minimum free energy, and global untwisting of forced supertwist is energetically favorable, consistent with our experimental data. Finally, we show that attachment of Cy3 dyes or changing counterions can cause nanotubes to adopt a permanent writhe with micrometer-scale pitch and amplitude. We propose that the coupling of local twist and global counter-twist may be useful in characterizing perturbations of DNA structure.

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Influenza is a severe disease in humans and animals with few effective therapies available. All strains of influenza virus are prone to developing drug resistance due to the high mutation rate in the viral genome. A therapeutic agent that targets a highly conserved region of the virus could bypass resistance and also be effective against multiple strains of influenza. Influenza uses many individually weak ligand binding interactions for a high avidity multivalent attachment to sialic acid-bearing cells. Polymerized sialic acid analogs can form multivalent interactions with influenza but are not ideal therapeutics due to solubility and toxicity issues. We used liposomes as a novel means for delivery of the glycan sialylneolacto-N-tetraose c (LSTc). LSTc-bearing decoy liposomes form multivalent, polymer-like interactions with influenza virus. Decoy liposomes competitively bind influenza virus in hemagglutination inhibition assays and inhibit infection of target cells in a dose-dependent manner. Inhibition is specific for influenza virus, as inhibition of Sendai virus and respiratory syncytial virus is not observed. In contrast, monovalent LSTc does not bind influenza virus or inhibit infectivity. LSTc decoy liposomes prevent the spread of influenza virus during multiple rounds of replication in vitro and extend survival of mice challenged with a lethal dose of virus. LSTc decoy liposomes co-localize with fluorescently tagged influenza virus, whereas control liposomes do not. Considering the conservation of the hemagglutinin binding pocket and the ability of decoy liposomes to form high avidity interactions with influenza hemagglutinin, our decoy liposomes have potential as a new therapeutic agent against emerging influenza strains.

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We show that DNA hairpins template the site-specific assembly of fluorescent few-atom Ag clusters on DNA nanotubes. Fluorescent clusters form only at hairpin sites and not on the double-stranded DNA scaffold, allowing for spatially programmed self-assembly. Ag clusters synthesized on hairpins protruding from DNA nanotubes can have nearly identical fluorescence spectra to those synthesized on free hairpins of identical sequence. Analysis of the stepwise photobleaching of individual clusters suggests a chemical yield of ~45%. Given the well-established sequence-specific optical properties of DNA stabilized Ag clusters, these results point the way toward high yield assembly of metal cluster fluorophores with control over spectra as well as spatial arrangement.

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Cells are capable of a variety of dramatic stimuli-responsive mechanical behaviors. These capabilities are enabled by the pervading cytoskeletal network, an active gel composed of structural filaments (e.g., actin) that are acted upon by motor proteins (e.g., myosin). Here, we describe the synthesis and characterization of an active gel using noncytoskeletal components. We use methods of base-pair-templated DNA self assembly to create a hybrid DNA gel containing stiff tubes and flexible linkers. We then activate the gel by adding the motor FtsK50C, a construct derived from the bacterial protein FtsK that, in vitro, has a strong and processive DNA contraction activity. The motors stiffen the gel and create stochastic contractile events that affect the positions of attached beads. We quantify the fluctuations of the beads and show that they are comparable both to measurements of cytoskeletal systems and to theoretical predictions for active gels. Thus, we present a DNA-based active gel whose behavior highlights the universal aspects of nonequilibrium, motor-driven networks.

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Among the key goals of structural DNA nanotechnology are to build highly ordered structures self-assembled from individual DNA motifs in 1D, 2D, and finally 3D. All three of these goals have been achieved with a variety of motifs. Here, we report the design and characterization of 1D nanotubes and 2D arrays assembled from three novel DNA motifs, the 6-helix bundle (6HB), the 6-helix bundle flanked by two helices in the same plane (6HB+2), and the 6-helix bundle flanked by three helices in a trigonal arrangement (6HB+3). Long DNA nanotubes have been assembled from all three motifs. Such nanotubes are likely to have applications in structural DNA nanotechnology, so it is important to characterize their physical properties. Prominent among these are their rigidities, described by their persistence lengths, which we report here. We find large persistence lengths in all species, around 1-5 µm. The magnitudes of the persistence lengths are clearly related to the designs of the linkages between the unit motifs. Both the 6HB+2 and the 6HB+3 motifs have been successfully used to produce well-ordered 2D periodic arrays via sticky-ended cohesion.

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We have investigated the formation of phospholipid bilayers of the neutral (zwitterionic) lipid dimyristoyl-phosphatidylcholine (DMPC) on various glass surfaces from vesicles in various aqueous solutions and temperatures using a number of complementary techniques: the surface forces apparatus (SFA), quartz crystal microbalance (QCM), fluorescence recovery after photobleaching (FRAP), fluorescence microscopy, and streaming potential (SP) measurements. The process involves five stages: vesicle adhesion to the substrate surfaces via electrostatic and van der Waals forces, steric interactions with neighboring vesicles, rupture, spreading via hydrophobic fusion of bilayer edges, and ejection of excess lipid, trapped water, and ions into the solution. The forces between DMPC bilayers and silica were measured in the SFA in phosphate buffered saline (PBS), and the adhesion energy was found to be 0.5-1 mJ/m(2) depending on the method of bilayer preparation. This value is stronger than the expected adhesion predicted by van der Waals interactions. Theoretical analysis of the bilayer-silica interaction shows that the strong attraction is likely due to an attractive electrostatic interaction between the uncharged bilayer and negatively charged silica owing to the surfaces interacting at "constant potential." However, the bilayer-silica interaction in distilled water was found to be repulsive at all distances, which is attributed to the surfaces interacting at "constant charge." These results are consistent with QCM measurements that show vesicles readily forming bilayers on silica in high salt but only weakly adhering in low salt conditions. We conclude that the electrostatic interaction is the most important interaction in determining the adhesion between neutral bilayers and charged hydrophilic surfaces. SP and FRAP experiments gave insights into the bilayer formation process as well as information on the surface coverage, lateral diffusion of the lipid molecules, and surface potential of the bilayers during the spreading process.

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Tau, a family of microtubule-associated proteins (MAPs), stabilizes microtubules (MTs) and regulates their dynamics. Tau isoforms regulate MT dynamic instability differently: 3-repeat tau is less effective than 4-repeat tau at suppressing the disassembly of MTs. Here, we report another tau-isoform-dependent phenomenon, revealed by fluorescence recovery after photobleaching measurements on a BODIPY-conjugated taxol bound to MTs. Saturating levels of recombinant full-length 3-repeat and 4-repeat tau both cause taxol mobility to be remarkably sensitive to taxol concentration. However, 3-repeat tau induces 2.5-fold faster recovery ( approximately 450s) at low taxol concentrations ( approximately 100 nM) than 4-repeat tau ( approximately 1000 s), indicating that 3-repeat tau decreases the probability of taxol rebinding to its site in the MT lumen. Finding no tau-induced change in the MT-binding affinity of taxol, we conclude that 3-repeat tau either competes for the taxol binding site with an affinity of approximately 1 microM or alters the MT structure so as to facilitate the passage of taxol through pores in the MT wall.

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DNA self-assembly provides a programmable bottom-up approach for the synthesis of complex structures from nanoscale components. Although nanotubes are a fundamental form encountered in tile-based DNA self-assembly, the factors governing tube structure remain poorly understood. Here we report and characterize a new type of nanotube made from DNA double-crossover molecules (DAE-E tiles). Unmodified tubes range from 7 to 20 nm in diameter (4 to 10 tiles in circumference), grow as long as 50 microm with a persistence length of approximately 4 microm, and can be programmed to display a variety of patterns. A survey of modifications (1) confirms the importance of sticky-end stacking, (2) confirms the identity of the inside and outside faces of the tubes, and (3) identifies features of the tiles that profoundly affect the size and morphology of the tubes. Supported by these results, nanotube structure is explained by a simple model based on the geometry and energetics of B-form DNA.

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We present the first direct observations of tile-based DNA self-assembly in solution using fluorescent nanotubes composed of a single tile. The nanotubes reach tens of microns in length by end-to-end joining rather than by sequential addition of single tiles. Their exponential length distributions withstand dilution but decay via scission upon heating, with an energy barrier Esc approximately 180kBT. DNA nanotubes are thus uniquely accessible equilibrium polymers that enable new approaches to optimizing DNA-based programming and understanding the biologically programmed self-assembly of protein polymers.

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The standard model for the structure of collagen in tendon is an ascending hierarchy of bundling. Collagen triple helices bundle into microfibrils, microfibrils bundle into subfibrils, and subfibrils bundle into fibrils, the basic structural unit of tendon. This model, developed primarily on the basis of x-ray diffraction results, is necessarily vague about the cross-sectional organization of fibrils and has led to the widespread assumption of laterally homogeneous closepacking. This assumption is inconsistent with data presented here. Using atomic force microscopy and micromanipulation, we observe how collagen fibrils from tendons behave mechanically as tubes. We conclude that the collagen fibril is an inhomogeneous structure composed of a relatively hard shell and a softer, less dense core.

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