Maik Hadorn

Specific and reversible DNA-directed self-assembly of oil-in-water emulsion droplets

Higher-order structures that originate from the specific and reversible DNA-directed self-assembly of microscopic building blocks hold great promise for future technologies. Here, we functionalized biotinylated soft colloid oil-in-water emulsion droplets with biotinylated single-stranded DNA oligonucleotides using streptavidin as an intermediary linker. We show the components of this modular linking system to be stable and to induce sequence-specific aggregation of binary mixtures of emulsion droplets. Three length scales were thereby involved: nanoscale DNA base pairing linking microscopic building blocks resulted in macroscopic aggregates visible to the naked eye. The aggregation process was reversible by changing the temperature and electrolyte concentration and by the addition of competing oligonucleotides. The system was reset and reused by subsequent refunctionalization of the emulsion droplets. DNA-directed self-assembly of oil-in-water emulsion droplets, therefore, offers a solid basis for programmable and recyclable soft materials that undergo structural rearrangements on demand and that range in application from information technology to medicine.

DNA-mediated self-assembly of artificial vesicles.

Background Although multicompartment systems made of single unilamellar vesicles offer the potential to outperform single compartment systems widely used in analytic, synthetic, and medical applications, their use has remained marginal to date. On the one hand, this can be attributed to the binary character of the majority of the current tethering protocols that impedes the implementation of real multicomponent or multifunctional systems. On the other hand, the few tethering protocols theoretically providing multicompartment systems composed of several distinct vesicle populations suffer from the readjustment of the vesicle formation procedure as well as from the loss of specificity of the linking mechanism over time. Methodology/Principal Findings In previous studies, we presented implementations of multicompartment systems and resolved the readjustment of the vesicle formation procedure as well as the loss of specificity by using linkers consisting of biotinylated DNA single strands that were anchored to phospholipid-grafted biotinylated PEG tethers via streptavidin as a connector. The systematic analysis presented herein provides evidences for the incorporation of phospholipid-grafted biotinylated PEG tethers to the vesicle membrane during vesicle formation, providing specific anchoring sites for the streptavidin loading of the vesicle membrane. Furthermore, DNA-mediated vesicle-vesicle self-assembly was found to be sequence-dependent and to depend on the presence of monovalent salts. Conclusions/Significance This study provides a solid basis for the implementation of multi-vesicle assemblies that may affect at least three distinct domains. (i) Analysis. Starting with a minimal system, the complexity of a bottom-up system is increased gradually facilitating the understanding of the components and their interaction. (ii) Synthesis. Consecutive reactions may be implemented in networks of vesicles that outperform current single compartment bioreactors in versatility and productivity. (iii) Personalized medicine. Transport and targeting of long-lived, pharmacologically inert prodrugs and their conversion to short-lived, active drug molecules directly at the site of action may be accomplished if multi-vesicle assemblies of predefined architecture are used.

Defined DNA-mediated assemblies of gene-expressing giant unilamellar vesicles

The technological aspects of artificial vesicles as prominent cell mimics are evolving toward higher-order assemblies of functional vesicles with tissuelike architectures. Here, we demonstrate the spatially controlled DNA-directed bottom-up synthesis of complex microassemblies and macroassemblies of giant unilamellar vesicles functionalized with a basic cellular machinery to express green fluorescent protein and specified neighbor-to-neighbor interactions. We show both that the local and programmable DNA pairing rules on the nanoscale are able to direct the microscale vesicles into macroscale soft matter assemblies and that the highly sensitive gene-expression machinery remains intact and active during multiple experimental steps. An in silico model recapitulates the experiments performed in vitro and covers additional experimental setups highlighting the parameters that control the DNA-directed bottom-up synthesis of higher-order self-assembled structures. The controlled assembly of a functional vesicle matrix may be useful not only as simplified natural tissue mimics but also as artificial scaffolds that could interact and support living cells.

Hierarchical Unilamellar Vesicles of Controlled Compositional Heterogeneity

Eukaryotic life contains hierarchical vesicular architectures (i.e. organelles) that are crucial for material production and trafficking, information storage and access, as well as energy production. In order to perform specific tasks, these compartments differ among each other in their membrane composition and their internal cargo and also differ from the cell membrane and the cytosol. Man-made structures that reproduce this nested architecture not only offer a deeper understanding of the functionalities and evolution of organelle-bearing eukaryotic life but also allow the engineering of novel biomimetic technologies. Here, we show the newly developed vesicle-in-water-in-oil emulsion transfer preparation technique to result in giant unilamellar vesicles internally compartmentalized by unilamellar vesicles of different membrane composition and internal cargo, i.e. hierarchical unilamellar vesicles of controlled compositional heterogeneity. The compartmentalized giant unilamellar vesicles were subsequently isolated by a separation step exploiting the heterogeneity of the membrane composition and the encapsulated cargo. Due to the controlled, efficient, and technically straightforward character of the new preparation technique, this study allows the hierarchical fabrication of compartmentalized giant unilamellar vesicles of controlled compositional heterogeneity and will ease the development of eukaryotic cell mimics that resemble their natural templates as well as the fabrication of novel multi-agent drug delivery systems for combination therapies and complex artificial microreactors.

Water Floating Self-assembling Agents

In this paper, we present a novel model of autonomous floating agents which can self-assemble on a water surface. We focus especially on the shape of the agent, which is essential as a factor of aggregation but difficult to deal with as finite states. After several experiments, a unique self-assembling behavior was observed by exploiting several physical-level forces; 6 agents gather into a cluster and form a specific pattern, which is unique to the configuration. This is considered crucial to achieve morphogenesis of a multi-agent system, of which we think as a hierarchical aggregation. The results shown here provide a good starting point for speculation about the level of autonomy of a agent in the complex environment when the system creates a self-assembling pattern.

The MATCHIT Automaton: Exploiting Compartmentalization for the Synthesis of Branched Polymers

We propose an automaton, a theoretical framework that demonstrates how to improve the yield of the synthesis of branched chemical polymer reactions. This is achieved by separating substeps of the path of synthesis into compartments. We use chemical containers (chemtainers) to carry the substances through a sequence of fixed successive compartments. We describe the automaton in mathematical terms and show how it can be configured automatically in order to synthesize a given branched polymer target. The algorithm we present finds an optimal path of synthesis in linear time. We discuss how the automaton models compartmentalized structures found in cells, such as the endoplasmic reticulum and the Golgi apparatus, and we show how this compartmentalization can be exploited for the synthesis of branched polymers such as oligosaccharides. Lastly, we show examples of artificial branched polymers and discuss how the automaton can be configured to synthesize them with maximal yield.

A Quantitative Analytical Method to Test for Salt Effects on Giant Unilamellar Vesicles

Today, free-standing membranes, i.e. liposomes and vesicles, are used in a multitude of applications, e.g. as drug delivery devices and artificial cell models. Because current laboratory techniques do not allow handling of large sample sizes, systematic and quantitative studies on the impact of different effectors, e.g. electrolytes, are limited. In this work, we evaluated the Hofmeister effects of ten alkali metal halides on giant unilamellar vesicles made of palmitoyloleoylphosphatidylcholine for a large sample size by combining the highly parallel water-in-oil emulsion transfer vesicle preparation method with automatic haemocytometry. We found that this new quantitative screening method is highly reliable and consistent with previously reported results. Thus, this method may provide a significant methodological advance in analysis of effects on free-standing model membranes.

Actuated bivalve robot study of the burrowing locomotion in sediment

This paper presents the design and control of an actuated bivalve robot, which has been developed to study the burrowing locomotion of bivalves in sediment. The setup consists of a tank filled with sand and water, plastic models of bivalve shells capable of expelling water and an external actuation mechanism simulating the rocking burrowing motion typically used by these animals. The realistic shell shapes have been realized using three-dimensional plotting techniques allowing testing influences of different shell shapes and surface structures (sculptures) on the burrowing efficiency. Based on the experimental setup, the burrowing process has been reproduced. The results show that this setup can be used to identify correlations in the burrowing process. Further experimental work will investigate the influence of factors such as shell shape and sculpture or the motion sequence on the burrowing performance.

Detection of Antibodies to the Feline Leukemia Virus (FeLV) Transmembrane Protein p15E: an Alternative Approach for Serological FeLV Detection Based on Antibodies to p15E

ABSTRACT The aim of this report was to investigate whether the diagnosis of feline leukemia virus (FeLV) infection by serology might be feasible and useful. Among the various viral proteins, the FeLV env-gene product (SU) and the envelope transmembrane protein p15E were considered promising candidates for the serological diagnosis of FeLV infection. Thus, we evaluated p15E and three other FeLV antigens, namely, a recombinant env-gene product, whole FeLV, and a short peptide from the FeLV transmembrane protein, for their potential to detect FeLV infection. To evaluate possible exposure of cats to FeLV, we tested serum and plasma samples from experimentally and naturally infected and vaccinated cats for the presence of antibodies to these antigens by enzyme-linked immunosorbent assays (ELISAs). The serological results were compared with the p27 and proviral real-time PCR results. We found that p15E displayed a diagnostic sensitivity of 95.7% and a specificity of 100% in experimentally infected cats. In naturally infected cats, p15E showed a diagnostic sensitivity of 77.1% and a specificity of 85.6%. Vaccinated cats displayed minimal antibody levels to p15E, suggesting that anti-p15E antibodies indicate infection rather than vaccination. The other antigens turned out to be too unspecific. The lower specificity in cats exposed to FeLV under field conditions may be explained by the fact that some cats become infected and seroconvert in the absence of detectable viral nucleic acids in plasma. We conclude that p15E serology may become a valuable tool for diagnosing FeLV infection; in some cases, it may replace PCR.

Towards Tailored Communication Networks in Assemblies of Artificial Cells

Living Technology is researching novel IT making strong use of programmable chemical systems. These chemical systems shall finally converge to artificial cells resulting in evolvable complex information systems. We focus on procedural manageability and information processing capabilities of such information systems. Here, we present a novel resource-saving formation, processing, and examination procedure to generate and handle single compartments representing preliminary stages of artificial cells. Its potential is exemplified by testing the influence of different glycerophospholipids on the stability of the compartments. We discuss how the procedure could be used both in evolutionary optimization of self-assembling amphiphilic systems and in engineering tailored communication networks enabling life-like information processing in multicompartment aggregates of programmable composition and spatial configuration.