DNA Nanostructures on Membranes as Tools for Synthetic Biology

Over the last decade, functionally designed DNA nanostructures applied to lipid membranes prompted important achievements in the fields of biophysics and synthetic biology. Taking advantage of the universal rules for self-assembly of complementary oligonucleotides, DNA has proven to be an extremely versatile biocompatible building material on the nanoscale. The possibility to chemically integrate functional groups into oligonucleotides, most notably with lipophilic anchors, enabled a widespread usage of DNA as a viable alternative to proteins with respect to functional activity on membranes. As described throughout this review, hybrid DNA-lipid nanostructures can mediate events such as vesicle docking and fusion, or selective partitioning of molecules into phase-separated membranes. Moreover, the major benefit of DNA structural constructs, such as DNA tiles and DNA origami, is the reproducibility and simplicity of their design. DNA nanotechnology can produce functional structures with subnanometer precision and allow for a tight control over their biochemical functionality, e.g., interaction partners. DNA-based membrane nanopores and origami structures able to assemble into two-dimensional networks on top of lipid bilayers are recent examples of the manifold of complex devices that can be achieved. In this review, we will shortly present some of the potentially most relevant avenues and accomplishments of membrane-anchored DNA nanostructures for investigating, engineering, and mimicking lipid membrane-related biophysical processes

Nonspecific Interactions of Amphiphilic Nanoparticles and Biomimetic Membranes

Robust control over nanoparticle (NP)-cell interactions is essential to the rational design of safe, advanced, and tailored nano-bio technologies. Achieving control over NP behavior at the cellular interface relies on the fundamental need to elucidate the physicochemical principles underlying the interactions between NPs and cell membranes. Despite numerous research efforts, the propensity of surface-modified NPs to interfere (or not) with the organization and function of cell membranes is still far from clear due to the inherently complex and dynamic nature of these biological barriers. In this thesis, experimental investigations were undertaken to tackle some aspects of nonspecific NP-membrane interactions that are still unclear or very poorly addressed. Sub-5 nm gold NPs protected by a mixture of hydrophobic and \u3c9-charged hydrophilic thiols were considered. These amphiphilic NPs possess high biomedical potential as they are able to passively enter living cells for theranostic purposes. Based on a biomimetic approach, lipid bilayers of varying structural and morphological complexity were employed to model the lipid structure of cell membranes. This work revealed that the sign of the NP surface charge is not responsible for different NP behavior in the interaction with neutral membranes. Notably, anionic and cationic NPs were shown not to damage the membrane integrity during passive bilayer penetration. Furthermore, anionic NPs were revealed to perturb the lateral lipid phase separation of multidomain membranes in a concentration-dependent manner and form peculiar bilayer-embedded ordered aggregates. Finally, the cholesterol-tuned reduction in bilayer fluidity was disclosed to dramatically hinder passive NP uptake into fluid membranes. Taken together, these findings provide a novel contribution in elucidating how amphiphilic entities endowed with surface conformational flexibility, such as ligand-protected NPs, can interact with cell membranes

PARL et HAX1 dans la régulation de l'activité mitochondriale

Haxl joue un rôle important dans les syndromes d'immunodéficience et l'apoptose. Une étude récente suggère que Haxl, une protéine membre de la famille Bcl-2, inhibe l'apoptose dans les neurones et les lymphocytes, via un mécanisme impliquant sa liaison avec PARL, la protease rhomboïde de la membrane mitochondriale, ce qui active par protéolyse la serine protease Omi/HtrA2 et élimine Haxl actif. Ce modèle indique que la sensibilité des cellules aux stimuli pro-apoptotiques est contrôlée par le complexe PARL/Haxl de l'espace intermembranaire de la mitochondrie. D'une manière plus globale, les protéines membres de la famille Bcl-2 pourraient contrôler la perméabilité de la membrane mitochondriale externe à partir de l'intérieur de la mitochondrie. De plus, ce modèle définit une nouvelle voie anti-apoptotique de PARL, indépendante de Opal. Dans la présente étude, nous montrons que, in vivo, l'activité de Haxl ne peut pas être couplée à PARL, car les deux protéines sont dans des compartiments cellulaires différents, et leur interaction in vitro est un artefact. Par une analyse de séquence et de prédiction de structure secondaire, nous montrons aussi que Haxl n'est pas membre de la famille Bcl-2, en raison de l'absence des modules d'homologie de Bcl-2. Ces résultats indiquent la présence de fonctions et de mécanismes différents de Haxl dans l'apoptose, et ouvrent de nouvelles questions sur la capacité de PARL de réguler, en plus de l'apoptose, le stress mitochondrial via une voie Omi/HtrA2 dépendante

Engineered core - shell nanoparticles: synthesis, characterisation, and biocompatibility studies

Superparamagnetic iron oxide nanoparticles (SPIONs) have emerged as promising contrast agents for Magnetic Resonance Imaging (MRI). Some SPIONs are already approved for clinical use. Coating of these nanoparticles with an additional biocompatible layer serves to improve the colloidal stability and biocompatibility. The present thesis is focused on the synthesis, characterization, and in vitro biocompatibility assessment of SPIONs as well as the assessment of the potential impact of the so-called bio-corona on the surface of these nanoparticles on their behavior. In addition, synthesis and magnetic evaluation of novel nanocomposites was also performed. In Paper I, the production of mono-dispersed, necking-free, single-core iron oxide-silica shell nanoparticles with tunable shell thickness was achieved by a carefully optimized inverse microemulsion method. The development of novel nanomaterials for biomedical applications need to be accompanied by careful scrutiny of their biocompatibility with a particular focus on the possible interactions with the primary defense system against foreign invasion, the immune system. Consequently, in Paper II, our efforts in synthesizing high quality core–shell nanoparticles with superparamagnetic character and sufficiently high magnetization were coupled with in vitro biocompatibility assessment and studies of cellular internalization using primary human macrophages and dendritic cells. The silica-coated nanoparticles were nontoxic to primary human monocyte-derived macrophages at all doses tested whereas dose- and size-dependent toxicity was observed for primary monocyte-derived dendritic cells exposed to smaller silica-coated nanoparticles, but not for the same-sized, commercially available dextran-coated iron oxide nanoparticles. Furthermore, the silica-coated iron oxide nanoparticles were taken up in both cell types through an active, actin cytoskeleton-dependent process to a significantly higher degree when compared to the dextran-coated nanoparticles, irrespective of size. This has potentially useful implications for labeling of immune cells for visualization, diagnosis or treatment of inflammatory processes. When nanomaterials confront physiological media, the formation of a “corona” of proteins by adsorption to the surface of nanomaterials occurs which will influence how the particles will interact with a biological system and consequently will affect the fate of the particles. The potential effect of the protein corona on the magnetic and biological behavior of silica- versus dextran-coated SPIONs was addressed in Paper III. A thorough physical characterization of SPIONs without and with a protein corona as well as in vitro biocompatibility and cellular internalization using human primary macrophages was performed. Modulation of the magnetization and relaxivity signals of the SPIONs was noted following interaction with human plasma proteins. Macrophage viability was influenced by the presence or absence of a protein corona on silica-coated SPIONs but in the case of the dextran-coated SPIONs. Macrophage production of pro-inflammatory TNF-α was not triggered by SPIONs with or without a corona. Moreover, comprehensive assessment of the protein corona using mass spectrometry and bioinformatics tools revealed distinctive compositions on the two types of nanoparticles. Further studies need to be performed to understand the interrelationship between the acquired protein corona on the SPIONs and their function as MRI contrast agents. In Paper IV, incorporation of iron oxide nanoparticles homogeneously dispersed in a polymeric matrix and assessment of the magnetic and optical properties of the resulting structured nanomaterials are presented. Magnetic evaluation of the nanocomposite revealed its ferromagnetic properties while the low loading of nanoparticles with very good distribution in the nanostructure yielded materials with good magneto-optical properties. These materials have potential applications as micro actuators, sensors, relays and magneto optical devices based on the Faraday effect. Overall, this thesis attempts an interdisciplinary approach to the synthesis and characterization of nanomaterials and their biocompatibility assessment, with the aim to enable future applications in nanomedicine

DNA PROGRAMMABLE SOFT MATTER DEVICES

The ability to program soft materials to undergo observable shape transformations in response to environmental stimuli is critical to the development soft programmable matter. In recent years, chemomechanical shape-changing hydrogels have garnered interest because they do not require wires or batteries and can operate untethered at smaller size scales. Devices comprised of these materials can respond to only a limited set of spatially non-specific stimuli such as temperature or pH - and are therefore restricted to a small set of final states. On the other hand, due to the large sequence space and programmable interactions of DNA molecules, devices comprised of DNA-conjugated hydrogel domains can potentially access a much larger set of final configurations through sequence-specific, addressable actuation of individual domains. To investigate the shape-changing properties of single domain DNA-conjugated hydrogels, we first determine the swelling extent of DNA-crosslinked acrylamide networks in response to sequence-specific DNA stimuli. By coupling the DNA crosslinks to a DNA hybridization chain reaction that enables further incorporation of DNA to the crosslink sites, we demonstrate that specific DNA molecules can induce up to 100-fold volumetric hydrogel expansion. This large degree of swelling is then used to actuate approximately centimeter-sized gels containing multiple DNA-sensitive gel domains that each change shape in response to different DNA sequences. From swelling experiments and finite-element simulations we develop a simple design rule for the DNA-controlled shape change of a hydrogel bilayer. The next generation of soft programmable matter and robotics will require materials that not only respond to distinct chemical species, but mechanical forces as well. Prior work in developing mechanochemically responsive polymers makes use of mechanophores - molecules that change configuration and initiate chemical reactions in response to mechanical forces - to instill bulk materials with force sensing properties. In this work, we use established thermodynamic models to design two DNA mechanophore complexes capable of responding to two distinct ranges of applied force. We micromold PEGDA copolymer hydrogels containing DNA mechanophore complexes and examine the force-sensing properties of the bulk material through the use of a multifunctional force microscope and a DNA-based fluorescence reporting scheme. Because DNA molecules can be coupled to molecular sensors, amplifiers, and logic circuits, the incorporation of DNA complexes into hydrogel networks - whether as mechanophores or chemical crosslinkers -introduces the possibility of building soft matter devices that respond to numerous, distinct inputs and autonomously implement chemical control programs. These soft matter constructs have the potential to exhibit the multistage, goal-directed behaviors that are currently impossible to achieve in other soft robotic devices

Biosynthesis of fungal polyketide natural products: Structural and biochemical studies toward understanding programming and substrate specificity

Polyketides are a large and diverse family of natural products encompassing some of the most effective and valuable pharmaceuticals of all time, as well as some of the deadliest toxins. These fascinating molecules are biosynthesized by some of nature’s largest and most complex enzymes, the polyketide synthases. Because of their diverse biological activities, polyketide-based natural products are an attractive target for biosynthetic engineering. In order to effectively engineer polyketide synthases to produce novel compounds, we must understand the native enzymology of these molecular machines. Since they consist of only a single set of catalytic domains, the fungal, iterative polyketide synthases are particularly challenging to decipher and engineer, relative to the assembly-line-like modular polyketide synthases. In this work, biochemical and structural studies are performed to improve understanding of iterative polyketide synthase enzymatic programming. By performing a screen of thirty unnatural starter units, the substrate specificity of the iterative polyketide synthase PksA was evaluated. Characterization of enzymatic derailment products allowed for interrogation of the tolerance of specific catalytic domains to the unnatural substrates. Through mechanism-based crosslinking of a closely related iterative polyketide synthase, CTB1, the structure of a polyketide synthase locked in a loading conformation was observed by cryo-electron microscopy. This first-ever multidomain structure of a non-reducing polyketide synthase may help to guide future engineering efforts, by beginning to establish what conformational changes polyketide synthases undergo during biosynthesis and how programming is achieved across multiple catalytic domains

Annual Report, 2013-2014

Beginning in 2004/2005- issued in online format onl

3D Bioprinted Engineered Living Materials for Continuous Organophosphorus Compound Detoxification

Engineered living materials (ELMs) are a rapidly emerging class of materials, demonstrating a wide range of functionalities, including responsive morphing, self-healing, and bio-catalysis. 3D bioprinted hydrogels have been used for the fabrication of high resolution, compartmentalised, and load-bearing structures suitable for hosting microbial metabolism, and accordingly represent an ideal environment for ELMs. The interactions between material frameworks, such as hydrogels, and encapsulated life are now beginning to be investigated.Herein, by 3D printing a hydrogel-encapsulated population of Escherichia coli, a chemically inducible, metabolically active, microbial ELM was fabricated. The material was characterised using a wide range of techniques, including fluorescence microscopy and cryogenic electron microscopy. Toxic organophosphorus compound (OPC) detoxifying capabilities were conveyed to the material through inducible expression of Agrobacterium radiobacter phosphotriesterase (arPTE). The reaction diffusion process occurring at the interface of the OPC detoxifying ELM was investigated using continuous fluorescence imaging of Coumaphos hydrolysis.. Principal component analysis was then used to uncover spatial and temporal features within this data, with relevance for future optimisation of catalytic microbial ELMstructures. To further demonstrate the applicability of this 3D printable microbial ELM, the material was incorporated into an entirely 3D printed flow reactor, demonstrating effective, cyclical detoxification of an OPC solution at high flow rate.Looking towards the future of ELM design, a novel, 3D printable, contractile-thermosensitive,double-network hydrogel was used to create thermo-responsive OPC degrading bioreactors, capable of autonomously controlling their performance