Accurate phase diagram of tetravalent DNA nanostars

We evaluate, by means of molecular dynamics simulations employing a realistic DNA coarse-grained model, the phase behaviour and the structural and dynamic properties of tetravalent DNA nanostars, i.e. nanoconstructs completely made of DNA. We find that, as the system is cooled down, tetramers undergo a gas--liquid phase separation in a region of concentrations which, if the difference in salt concentration is taken into account, is comparable with the recently measured experimental phase diagram [S. Biffi \textit{et al}, Proc. Natl. Acad. Sci, \textbf{110}, 15633 (2013)]. We also present a mean-field free energy for modelling the phase diagram based on the bonding contribution, derived by Wertheim in its studies of associating liquids, combined with mass action law expressions appropriate for DNA binding and a numerically evaluated reference free energy. The resulting free energy qualitatively reproduces the numerical data. Finally, we report information on the nanostar structure, e.g. geometry and flexibility of the single tetramer and of the collective behaviour, providing a useful reference for future small angle scattering experiments, for all investigated temperatures and concentrations.Comment: 10 pages, 11 figure

Correction: Equilibrium gels of low-valence DNA nanostars: a colloidal model for strong glass formers

n/

Small-angle neutron scattering and Molecular Dynamics structural study of gelling DNA nanostars

DNA oligomers with properly designed sequences self-assemble into well defined constructs. Here, we exploit this methodology to produce bulk quantities of tetravalent DNA nanostars (each one composed by 196 nucleotides) and to explore the structural signatures of their aggregation process. We report small-angle neutron scattering experiments focused on the evaluation of both the form factor and the temperature evolution of the scattered intensity at a nano star concentration where the system forms a tetravalent equilibrium gel. We also perform molecular dynamics simulations of one isolated tetramer to evaluate the form factor theoretically, without resorting to any approximate shape. The numerical form factor is found to be in very good agreement with the experimental one. Simulations predict an essentially temperature independent form factor, offering the possibility to extract the effective structure factor and its evolution during the equilibrium gelation.Comment: 9 pages, 5 figure

Apparatus for simultaneous DLS-SANS investigations of dynamics and structure in soft matter

Dynamic Light Scattering (DLS) and Small-Angle Neutron Scattering (SANS) are two key tools with which to probe the dynamic and static structure factor, respectively, in soft matter. Usually DLS and SANS measurements are performed separately, in different laboratories, on different samples and at different times. However, this methodology has particular disadvantages for a large variety of soft materials which exhibit high sensitivity to small changes in fundamental parameters such as waiting times, concentration, pH, ionic strength, etc. Here we report on a new portable DLS-SANS apparatus that allows one to simultaneously measure both the microscopic dynamics (through DLS) and the static structure (through SANS) on the same sample. The apparatus has been constructed as a collaboration between two laboratories, each an expert in one of the scattering methods, and was commissioned on the \textit{LOQ} and \textit{ZOOM} SANS instruments at the ISIS Pulsed Neutron \& Muon Source, U.K

Experimental study of the phase behaviour of limited valence particles in DNA nano-aggregate systems

In the last years, we have witnessed to the unprecedented development of structural DNA nanotechnology, an innovative field of science based on the remarkable ability of DNA to hybridize in a highly specific and thermo-reversible fashion. Indeed, after the pioneering work of N. Seeman in 1982, there has been a progressive shift from the classical vision of DNA as a gene-encoding molecule of primary importance from biological perspectives to a much more innovative point of view, regarding DNA as a building block of considerable nanotechnological relevance. In particular, recent progress in DNA synthesis and manipulation have made it possible to produce a large variety of DNA-based materials, including hydrogels, 2D and 3D crystals as well as more complex mesoscopic and macroscopic structures of various forms and functionalities. In the present thesis, following the lines set by structural DNA nanotechnology, we introduce a new twist: DNA nanoconstructs, i.e. nano-sized supramolecules entirely made of DNA, as man-designed particles to experimentally investigate unconventional phase behaviours conceived so far only in charta and in silico. In our view, DNA can be seen as a powerful tool to explore statistical physics because it enables to produce, via self-assembly, bulk quantities of identical particles with controlled mutual interactions. As a proof of concept, we focused on the phase behaviour of limited-valence particles (i.e. colloidal particles with small coordination numbers), a topic which has recently received a considerable interest, but which has so far been confined to theoretical and numerical investigations. Such investigations have shown that a solution of these particles should exhibit phase coexistence, the colloidal counterpart of the gas-liquid coexistence in simple liquids. The location of the unstable region in the temperature-concentration plane is predicted to be affected by the valence f, i.e. by the number of bonds that each particle can form. Specifically, the reduction of valence should lower both the critical temperature and the critical concentration, thus shrinking the coexistence region. However, despite such findings, the lack of a methodology for creating bulk quantities of particles with controlled valence has until now hindered the experimental investigation of the systematic dependence of the coexistence region on the valence. Therefore, we exploited the selectivity of DNA binding to realize soft particles as well as to control the inter-particle interactions. Specifically, in collaboration with the research group of Prof. T. Bellini (Department of Medical Biotechnology and Translational Medicine, University of Milan) we realized star-shaped DNA structures (nanostars) having three or four double-helical arms, each one ending with a sticky single-strand overhang designed on purpose to provide controllable and reversible interactions between individual structures. Therefore, in our view, such nanostars can be considered as limited-valence particles, whose valence is determined by the number of the arms. In the present thesis, we specifically addressed the f = 3 case, investigating the collective behaviour of trivalent DNA nanostars in a wide range of temperatures and densities. As expected, we found that solutions of such particles undergo phase separation. From a comparitive analysis with the results on f = 4 nanostar solutions, we discovered that, according to the expectations, the critical parameters crucially depend on the valence, causing a significant shrinking of the coexistence region as the valence is reduced. Moreover, we also characterized the critical dynamics of the system, finding a surprising anomalous behaviour. Indeed, upon approaching the critical point from high temperature, the scattered light intensity diverges with a power-law, whereas the field autocorrelation function shows a plateau followed by a slow relaxation process. Interstingly, the slow relaxation time exhibits an Arrhenius behaviour with no signs of criticality, demonstrating a novel scenario where the critical slowing down of the concentration fluctuations is enslaved to the large lifetime of the sticky bonds. Besides offering the chance of realizing bulk quantities of particles with low valence, our approach also provides a strong control over their mutual interactions, since the cohesion between sticky terminals can be easily tuned by changing the temperature of the system. Hence, the possibility of regulating the valence of the particles together with the chance of finely tuning their interactions in a reversible fashion provided the opportunity of investigating novel scenarios, such as equilibrium gelation processes. Specifically, once determined the phase diagram for f = 3 nanostars, and thus located the region of instability at low temperatures, we systematically investigated the dynamics of equilibrium gels and its dependence on the ionic strength of the medium. On approaching the gel state from high temperatures, we found that the intensity scattered by the solutions of f = 3 nanostars initially grows on cooling and it later saturates on a temperature-indipendent value, consistently with the expectations for equilibrium gels. Indeed, at sufficiently low temperatures, where the system reaches its fully bonded configuration, all nanostars are part of the same spanning infinite cluster and the topology of the network does not evolve anymore. Moreover, we found that, on gradually adding salt, the lifetime of the sticky bonds between nanostars decreases, progressively anticipating the decay of the density correlation functions. Thus, our results show that it is possible to tune the bond lifetime (and hence the dynamic behaviour of the system) by simply varying the ionic strength of the medium

Cold-swappable DNA gels

We report an experimental investigation of an all-DNA gel composed by tetra-functional DNA nanoparticles acting as network nodes and bi-functional ones acting as links. The DNA binding sequence is designed to generate at room and lower temperatures a persistent long-lived network. Exploiting ideas from DNA-nanotechnology, we implement in the binding base sequences an appropriate exchange reaction which allows links to swap, constantly retaining the total number of network links. The DNA gel is thus able to rearrange its topology at low temperature while preserving its fully-bonded configuration

Lysozyme binds onto functionalized carbon nanotubes

Single walled carbon nanotubes have singular physicochemical properties making them attractive in a wide range of applications. Studies on carbon nanotubes and biological macromolecules exist in literature. However, ad hoc investigations are helpful to better understand the interaction mechanisms. We report on a system consisting of single walled carbon nanotubes and lysozyme. The phenomenology of nanotube-protein interactions and its effects on protein conformation were determined. We investigated the formation of oxidized nanotube-lysozyme conjugates, by studying the effect of both protein concentration and pH. Electrophoretic mobility, dielectric spectroscopy and dynamic light scattering were used to determine the interaction pathways, monitoring the surface charge density and the size of the complexes. The results allowed identifying the conditions of surface saturation at different pH values. The secondary structure of nanotube-adsorbed protein was controlled by circular dichroism; it was observed that it substantially retains its native conformation. Interestingly, we found that the interactions among oxidized nanotubes and lysozyme molecules are mainly of electrostatic nature and easily tunable by varying the pH of the solutions. (C) 2013 Elsevier B.V. All rights reserved

Equilibrium gels of trivalent DNA-nanostars: Effect of the ionic strength on the dynamics

Abstract: Self-assembling DNA-nanostars are ideal candidates to explore equilibrium gelation in systems composed of limited-valence particles. We present here a light scattering study of the dynamics in a trivalent DNA-nanostars equilibrium gel and of its dependence on ionic strength and concentration. Reversible bonds between different nanostars, whose formation is sensitively dependent on temperature, concentration and ionic strength, are provided by complementary DNA sticky ends. We find that the decay of the density correlations is described by a two-step relaxation process characterised by: i) a slow time scale that varies over nearly four orders of magnitude in a temperature window of less than 30 degrees; ii) an increase of the amplitude (the so-called non-ergodicity factor) of the slow relaxation. The slow process follows an Arrhenius law with temperature. We observe that the activation enthalpy does not depend on the ionic strength and that the dependence of the relaxation time on the ionic strength can be rationalized in terms of the free-energy cost of forming a sticky-end duplex. Finally, we observe that dynamics is insensitive to nanostar concentration, in full agreement with the predicted behaviour in equilibrium gels. Graphical abstract: [Figure not available: see fulltext.

Phase behavior and critical activated dynamics of limited-valence DNA nanostars

Colloidal particles with directional interactions are key in the realization of new colloidal materials with possibly unconventional phase behaviors. Here we exploit DNA self-assembly to produce bulk quantities of "DNA stars" with three or four sticky terminals, mimicking molecules with controlled limited valence. Solutions of such molecules exhibit a consolution curve with an upper critical point, whose temperature and concentration decrease with the valence. Upon approaching the critical point from high temperature, the intensity of the scattered light diverges with a power law, whereas the intensity time autocorrelation functions show a surprising two-step relaxation, somehow reminiscent of glassy materials. The slow relaxation time exhibits an Arrhenius behavior with no signs of criticality, demonstrating a unique scenario where the critical slowing down of the concentration fluctuations is subordinate to the large lifetime of the DNA bonds, with relevant analogies to critical dynamics in polymer solutions. The combination of equilibrium and dynamic behavior of DNA nanostars demonstrates the potential of DNA molecules in diversifying the pathways toward collective properties and self-assembled materials, beyond the range of phenomena accessible with ordinary molecular fluids

Dietary Starch Concentration Affects Dairy Sheep and Goat Performances Differently during Mid-Lactation

Evolution of milk production, body reserves and blood metabolites and their relationships with dietary carbohydrates were compared in 30 Sarda dairy ewes and 26 Saanen dairy goats in mid-lactation. From 92 to 152 ± 11 days in milk (DIM), each species was allocated to two dietary treatments: high-starch (HS: 20.0% starch, on DM basis) and low-starch (LS: 7.8% starch, on DM basis) diets. In mid-lactating goats, the HS diet increased fat-corrected milk yield (FCM (3.5%); 2.65 vs. 2.53 kg/d; p = 0.019) and daily milk net energy (NEL; p = 0.025), compared to the LS diet. The body condition score (BCS) was not affected. In mid-lactating ewes, the LS diet increased FCM (6.5%) (1.47 vs. 1.36 kg/d; p = 0.008), and NEL (p = 0.008), compared to the HS diet. In addition, BCS was greater in HS than in LS ewes (3.53 vs. 3.38; p = 0.008). Goats had a higher growth hormone (GH) and lower insulin concentration than ewes (GH: 2.62 vs. 1.37 ng/mL; p = 0.04; insulin: 0.14 vs. 0.38 µg/L; p < 0.001 in goats and ewes, respectively). In conclusion, in mid-lactation, the two species responded differently to dietary carbohydrates, probably due to differences in the concentration of GH and insulin. The HS diet favored milk yield in goats and body reserve accumulation in ewes. In ewes, the partial replacement of starch with highly digestible fiber increased energy partitioning in favor of milk production