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

Abstract

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.