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Organelles of eukaryotic cells are structures made up of membranes, which carry out a majority of functions necessary for the surviving of the cell itself. Organelles also differentiate the prokaryotic and eukaryotic cells, and are arranged to form different compartments guaranteeing the activities for which eukaryotic cells are programmed. Cell membranes, containing organelles, are isolated from cancer cells and erythrocytes and used to form biocompatible and long circulating ghost nanoparticles delivering payloads or catalyzing enzymatic reactions as nanoreactors. In this attempt, red blood cell membranes were isolated from erythrocytes, and engineered to form nanoerythrosomes (NERs) of 150 nm. The horseradish peroxidase, used as an enzyme model, was loaded inside the aqueous compartment of NERs, and its catalytic reaction with Resorufm was monitored. The resulting nanoreactor protected the enzyme from proteolytic degradation, and potentiated the enzymatic reaction in situ as demonstrated by maximal velocity (V-max) and Michaelis constant (K-m), thus suggesting the high catalytic activity of nanoreactors compared to the pure enzymes.

Constructing higher-order vesicle assemblies has discipline-spanning potential from responsive soft-matter materials to artificial cell networks in synthetic biology. This potential is ultimately derived from the ability to compartmentalise and order chemical species in space. To unlock such applications, spatial organisation of vesicles in relation to one another must be controlled, and techniques to deliver cargo to compartments developed. Herein, we use optical tweezers to assemble, reconfigure and dismantle networks of cell-sized vesicles that, in different experimental scenarios, we engineer to exhibit several interesting properties. Vesicles are connected through double-bilayer junctions formed via electrostatically controlled adhesion. Chemically distinct vesicles are linked across length scales, from several nanometres to hundreds of micrometres, by axon-like tethers. In the former regime, patterning membranes with proteins and nanoparticles facilitates material exchange between compartments and enables laser-triggered vesicle merging. This allows us to mix and dilute content, and to initiate protein expression by delivering biomolecular reaction components., Assembly of higher-order artificial vesicles can unlock new applications. Here, the authors use optical tweezers to construct user-defined 2D and 3D architectures of chemically distinct vesicles and demonstrate inter-vesicle communication and light-enabled compartment merging.

The goal of my research program is to develop a DNA-based nanosensor for nucleic acids analysis. I plan to use DNA Origami nanostructures that are formed by a-few-thousand-nucleotides-long, circular, single stranded (ss)DNA “scaffold” folded to form a specific shape by the action of a few hundreds of short (approx. 30 nucleotides) ssDNA “staples”, which hybridize over non-consecutive regions of the scaffold. Staples can be incorporated within the structure with well-defined stoichiometry and some of them can be designed to serve as highly-specific receptor for short nucleic acids sequences. I plan to introduce a restriction site within staples adjacent to such probes to permit their steric protection from enzymatic degradation as a consequence of a probe-target recognition event in their vicinity. The restriction reaction, therefore, “writes” the amount of target molecules captured on the nanosensors by permanently modifying certain target-specific staples within the DNA nanostructure. In turn, the amount of such modified staples is associated with the amount of target molecules captured on the nanosensor surface from the solution, and can be subsequently analyzed with standard DNA quantification techniques such as quantitative PCR (qPCR), or high-throughput DNA sequencing. This research plan is based on recent results obtained in our laboratory showing that, in self-assembled DNA structures, restriction enzymatic reactions are steric-regulated in a step-wise fashion. Therefore, one goal of this PhD thesis is the development of a quantitative assay to evaluate the efficiency of enzymatic reactions within such nanostructures, as well as staples incorporation and stability for aiding fundamental studies of enzymatic reactions in DNA nanostructures. Specifically, the restriction quantification method proposed is based on a linear PCR (L-PCR) amplification reaction that involves staple-specific carriers (70-nucleotide-long ssDNA) that can fully hybridize to staple fragments produced by the enzymatic cutting. The polymerase action leads to the formation of a duplex DNA fragment 70 base pairs (bp) long for each cleaved staple, whereas the hybridization of un-cleaved staples on the carrier prevents such polymerase reaction. To study staples incorporation efficiency, the same protocol can be used, but DNA carriers are designed to hybridize the full length of the DNA staple sequence. I prepared L-PCR samples to evaluate next-generation sequencing (NGS) quantification accuracy and L-PCR efficiency. As a proof-of-concept, I analyzed 5 staples of a triangular DNA nanostructure and obtained information on their incorporation efficiency or cleavage. This thesis also describes the design and development of a DNA based nanosensor for improving the accuracy of short nucleic acid quantification with qPCR. The work aims at coupling qPCR with a self-assembled nanosensor, which can help overcome amplification and retro-transcription reaction bias, and circumvent the detection threshold of 2-fold concentration variation, without requiring updates to traditional qPCR instrumentation. Components of such sensor are three consecutive “foot-loop” DNA probes each carrying a target-complementary sequence in the loop. Probes are assembled over a common scaffold that joins their “feet”. Each “foot” carries a restriction site and upon hybridization of three copies of the same target molecule on the respective loops, the site of each foot is destabilized (termed “bingo” configuration). Only in this case, the whole scaffold is protected from enzymatic cleavage and can be amplified with PCR. Target and bingo-scaffold concentrations are correlated by power function of 3. The results obtained demonstrate the increased accuracy of the Bingo-qPCR assay with respect to standard qPCR in evaluating small variation of enzymatic activity, and prove the feasibility of the target detection switch-based reaction.

Lipases from Rhizomucor miehei (RmL) and Candida antartica (CaL-B) entrapped in surfactant free microemulsion-like ternary systems consisting of n-hexane, short chain alcohols (1-propanol or 2-methyl-2-propanol) and water, keep their catalytic activity at 30 degrees C, catalyzing efficiently the esterification of fatty acids or natural phenolic acids including cinnamic acid derivatives. High conversions (up to 95%) were obtained with both lipases entrapped in various ternary systems. Lipases catalytic efficiency and stability was influenced by the composition of the ternary systems used. Hydroxypropylmethyl cellulose (HPMC) microemulsions-based organogels (MBGs) formulated with various surfactant free microemulsion-like systems were used as a matrix for enzyme immobilization. These lipase-containing MBGs prove to be a novel solid-phase catalyst for use, under mild conditions, in non-polar solvents as well as in solvent free systems. CaL-B immobilized in surfactant free MBGs keep its catalytic activity for a storage period of 8 months, while the corresponding residual activity of immobilized RmL was decreased by 50%. The stability of enzymes in such surfactant free MBGs is higher than that observed in MBGs formulated with surfactant based w/o microemulsions. Lipases-containing surfactant free MBGs were used in seven consecutive batch syntheses of fatty esters with only 10-15% loss of activity. (c) 2005 Elsevier Inc. All rights reserved. Enzyme and Microbial Technology

Water-in-ionic liquid (w/IL) microemulsions formulated with non-ionic surfactants, (Tween 20 or Triton X-100) in 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim]PF(6)), were used as media for lipase-catalyzed esterification reactions. The catalytic behavior and stability of lipases from Candida rugosa, Chromobacterium viscosum and Thermomyces lanuginosa in these novel microemulsions were investigated and compared to other microheterogeneous media used so far for enzyme-catalyzed reactions. The catalytic behavior of the enzymes depends strongly on the surfactant concentration and the water content. The dependence of the esterification activity of lipases on molar ratio of water to surfactant (w(o)) follows a bell-shaped profile, presenting a maximum at w(o) approximate to 5. The operational stability of lipases in w/IL microemulsions, especially at high incubation temperature (50 degrees C), was significantly increased compared to that observed in other microheterogeneous media. The highest half-life times (>100 h) were obtained in w/IL microemulsions with low water content. Conformational studies via Fourier transform-infrared (FT-IR) and circular dichroism (CD) spectroscopy indicated that lipases entrapped in w/IL microemulsions in most cases retain their native structure or adapt a more rigid structure compared to other microheterogeneous media, which correlated well with the stability results. A simple procedure suitable for ester separation and enzyme reuse was developed. T lanuginosa lipase retained 90% of activity after ten reaction cycles in w/IL microemulsions formulated with Tween 20. (C) 2009 Elsevier B.V. All rights reserved. Journal of Molecular Catalysis B-Enzymatic

Surfactant free ternary systems formulated with organic solvents such as alpha-pinene or n-hexane, short chain alcohols and water, were used as new reaction media to maximize the catalytic performance of chloroperoxidase from Caldariomyces fumago (CPO) towards the oxidation of hydrophobic compounds including olefins and terpenols. The influence of the composition and the hydration state of the system, the nature of the oxidizing agent as well as substrate availability on the catalytic behavior and stability of the enzyme was investigated, using the chlorination of monochlorodimedone (MCD) and the epoxidation of styrene as model reactions. Based on residual chlorination activity measurements, which were in accordance with differential scanning calorimetry (DSC) findings, it was concluded that in low water systems formulated with alpha-pinene CPO stability is significantly increased compared to other reaction media (up to 65% residual activity after 24 h of incubation at 30 degrees C). Electron paramagnetic resonance (EPR) spectroscopy indicated that in low water content systems the ferric environment of the active site of CPO does not undergo severe modifications. Total turnover numbers (TTN) obtained for the oxidation of various olefins and terpenols in alpha-pinene-based systems, were up to 25,000 which is 2-170 times higher than previously reported values. The results indicate that such ternary systems can be efficiently employed as reaction media leading to highly enhanced stability and increase CPO potential as practical catalyst for oxidation of large amounts of hydrophobic compounds in a single-step biocatalytic process. (c) 2007 Elsevier B.V. All rights reserved. Journal of Molecular Catalysis B-Enzymatic

Modeling methods allow the identification and analysis of determinants of reactivity and specificity in enzymes. The reaction between glutathione and 1-chloro-2,4-dinitrobenzene (CDNB) is widely used as a standard activity assay for glutathione S-transferases (GSTs). It is important to understand the causes of differences between catalytic GST isoenzymes and the effects of mutations and genetic polymorphisms. Quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations have been performed here to investigate the addition of the glutathione anion to CDNB in the wild-type M1-1 GST isoenzyme from rat and in three single point mutant (Tyr6Phe, Tyr115Phe, and Met108Ala) M1-1 GST enzymes. We have developed a specifically parameterized QM/MM method (AM1-SRP/CHARMM22) to model this reaction by fitting to experimental heats of formation and ionization potentials. Free energy profiles were obtained from molecular dynamics simulations of the reaction using umbrella sampling and weighted histogram analysis techniques. The reaction in solution has also been simulated and is compared to the enzymatic reaction. The free energies are in excellent agreement with experimental results. Overall the results of the present study show that QM/MM reaction pathway analysis provides detailed insight into the chemistry of GST and can be used to obtain mechanistic insight into the effects of specific mutations on this catalytic process.