Reversible assembly of stacked membrane nanodiscs with reduced dimensionality and variable periodicity.

We demonstrate the self-organization of quasi-one-dimensional nanostructures with periodic features using nature's primary three building blocks: lipids, DNA, and proteins. The periodicity of these "BioNanoStacks" is controllable through selection of the length of the DNA spacers. We show that BioNanoStacks can be reversibly assembled and disassembled through thermal melting of the DNA duplex, where the melting transition temperature is controllable not just by the DNA sequence and salt concentration, but also by the lipid composition within these superstructures. These novel materials may find applications in fields such as templated nanomaterial assembly, tissue-engineering scaffolds, or therapeutic delivery systems. Well-established techniques for chemical modification of biomolecules will also provide a broad platform for adaption and remodeling of these structures to provide optimal features for the required application.

Using DNA-driven assembled phospholipid nanodiscs as a scaffold for gold nanoparticle patterning.

Recently, a new class of materials emerged with the assembly of DNA-coated phospholipid nanodiscs into columnar BioNanoStacks. Within these stacks, lipid discs are periodically incorporated, resulting into quasi-one-dimensional superstructures. With each disc surrounded by two recombinant scaffolding proteins, we decided to examine whether the polyhistidine tags of these proteins could be utilized to bind additional molecules or particles to these BioNanoStacks. Here we demonstrate that patterning of gold nanoparticles onto these BioNanoStacks is indeed possible. Binding occurs via a nickel-mediated interaction between the nanogolds nitrilotriacetic acid and the histidine tags of the scaffold proteins surrounding the nanodiscs. Using Monte Carlo simulations, we determine that the binding of the nanogold particles to the stacks is not a random event. By comparing the simulation and experimental results, we find that there are preferred binding sites, which affects the binding statistics.

Single vesicle observations of the cardiolipin-cytochrome C interaction: induction of membrane morphology changes.

We present a novel platform for investigating the composition-specific interactions of proteins (or other biologically relevant molecules) with model membranes composed of compositionally distinct domains. We focus on the interaction between a mitochondrial-specific lipid, cardiolipin (CL), and a peripheral membrane protein, cytochrome c (cyt c). We engineer vesicles with compositions such that they phase separate into coexisting liquid phases and the lipid of interest, CL, preferentially localizes into one of the domains (the liquid disordered (L(d)) phase). The presence of CL-rich and CL-depleted domains within the same vesicle provides a built-in control experiment to simultaneously observe the behavior of two membrane compositions under identical conditions. We find that cyt c binds strongly to CL-rich domains and observe fascinating morphological transitions within these regions of membrane. CL-rich domains start to form small buds and eventually fold up into a collapsed state. We also observe that cyt c can induce a strong attraction between the CL-rich domains of adjacent vesicles as demonstrated by the development of large osculating regions between these domains. Qualitatively similar behavior is observed when other polycationic proteins or polymers of a similar size and net charge are used instead of cyt c. We argue that these striking phenomena can be simply understood by consideration of colloidal forces between the protein and the membrane. We discuss the possible biological implications of our observations in relation to the structure and function of mitochondria.

DNA-mediated two-dimensional colloidal crystallization above different attractive surfaces.

We explore the formation of "floating" two-dimensional colloidal crystals above weakly attractive surfaces that are either positively or negatively charged. In particular, we studied crystal formation above positively charged poly-L-lysine-poly(ethylene glycol) surfaces with and without short single-stranded DNA and above negatively charged bovine albumin serum-streptavidin multilayers. Confocal microscopy revealed the evolution of crystals several micrometers above all three surfaces. Interestingly, the "flying height" of crystals was found to depend on the surface coating. All crystalline structures remained remarkably stable over weeks, even under high salt conditions. Neither lifting the crystals nor lowering them by means of buoyancy forces destroyed them.

A finite-cluster phase in λ-DNA-coated colloids.

We studied the aggregation of 1 µm colloids bridged by DNA with 32 µm contour length. We mixed two species of particles with grafted double-stranded λ-DNA displaying short, complementary single-stranded 'overhangs' as free binding-ends. Confocal microscopy showed the formation of stable, size-limited clusters in which the two species of particles were at touching contact. Simulations suggest that the observed close contact and the limitation to grow both result from entropic exclusion of the bridging DNA from the space between nearby particle surfaces.

Direct observation of size fractionation during colloidal crystallization.

We present a confocal microscopy study of the quasi-two-dimensional crystallization of a binary mixture of spherical colloids coated with long DNA strands. Our experiments show that in the crystalline phase the two colloidal species are completely demixed. Analysis of the lattice spacings in the two types of colloidal crystal shows that the diameters of the two species of colloids differ by 10%. We argue that the demixing in the crystalline phase is due to size segregation during crystallization. This phenomenon had been predicted in several theoretical studies. To our knowledge, the present study provides the first 'real-space' experimental confirmation of this effect.

Clustering versus percolation in the assembly of colloids coated with long DNA.

We report an experimental study in which we compare the self-assembly of 1 mum colloids bridged through hybridization of complementary single-stranded DNA (ssDNA) strands (12 bp) attached to variable-length double-stranded DNA spacers that are grafted to the colloids. We considered three different spacer lengths: long spacers (48 500 bp), intermediate length spacers (7500 bp), and no spacers (in which case the ssDNA strands were directly grafted to the colloids). In all three cases, the same ssDNA pairs were used. However, confocal microscopy revealed that the aggregation behavior is very different. Upon cooling, the colloids coated with short and intermediate length DNAs undergo a phase transition to a dense amorphous phase that undergoes structural arrest shortly after percolation. In contrast, the colloids coated with the longest DNA systematically form finite-sized clusters. We speculate that the difference is due to the fact that very long DNA can easily be stretched by the amount needed to make only intracluster bonds, and in contrast, colloids coated with shorter DNA always contain free binding sites on the outside of a cluster. The grafting density of the DNA decreases strongly with increasing spacer length. This is reflected in a difference in the temperature dependence of the aggregates: for the two systems coated with long DNA, the resulting aggregates were stable against heating, whereas the colloids coated with ssDNA alone would dissociate upon heating.