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Several strategies were investigated to address the three previously mentioned limitations. The first attempt was to study the effect length and conformation of polyethylene glycol (PEG) on DN stability. DNs were also coated with PEG-lipid and human serum albumin (HSA) and their stealth efficiencies were compared. The findings reveal that both PEGylation and albumin coating enhance low salt stability, increase resistance towards nuclease action and reduce uptake of DNs by macrophages. Any protective coating around a DN increases its hydrodynamic radius, which is a crucial parameter influencing their clearance. Keeping this in mind, intrinsically stable DNs that can survive low salt concentration without any polymer coating were built. Several DNA compaction agents and DNA binders were screened to stabilize DNs in low magnesium conditions. Among them arginine, lysine, bis-lysine and hexamine cobalt showed the potential to enhance DN stability.
This thesis also presents a sensitive assay, the Proximity Ligation Assay (PLA), for the estimation of DN stability with time. It requires very simple modifications on the DNs and it can yield precise results from a very small amount of sample. The applicability of PLA was successfully tested on several DNs ranging from a simple wireframe tetrahedron to a 3D origami and the protocol to collect in vivo samples, isolate the DNs and measure their stability was developed.
Swinging arms are a key functional component of multistep catalytic transformations in many naturally occurring multi-enzyme complexes. This arm is typically a prosthetic chemical group that is covalently attached to the enzyme complex via a flexible linker, allowing the direct transfer of substrate molecules between multiple active sites within the complex. Mimicking this method of substrate channelling outside the cellular environment requires precise control over the spatial parameters of the individual components within the assembled complex. DNA nanostructures can be used to organize functional molecules with nanoscale precision and can also provide nanomechanical control. Until now, protein–DNA assemblies have been used to organize cascades of enzymatic reactions by controlling the relative distance and orientation of enzymatic components or by facilitating the interface between enzymes/cofactors and electrode surfaces. Here, we show that a DNA nanostructure can be used to create a multi-enzyme complex in which an artificial swinging arm facilitates hydride transfer between two coupled dehydrogenases. By exploiting the programmability of DNA nanostructures, key parameters including position, stoichiometry and inter-enzyme distance can be manipulated for optimal activity.
A structurally and compositionally well-defined and spectrally tunable artificial light-harvesting system has been constructed in which multiple organic dyes attached to a three-arm-DNA nanostructure serve as an antenna conjugated to a photosynthetic reaction center isolated from Rhodobacter sphaeroides 2.4.1. The light energy absorbed by the dye molecules is transferred to the reaction center, where charge separation takes place. The average number of DNA three-arm junctions per reaction center was tuned from 0.75 to 2.35. This DNA-templated multichromophore system serves as a modular light-harvesting antenna that is capable of being optimized for its spectral properties, energy transfer efficiency, and photostability, allowing one to adjust both the size and spectrum of the resulting structures. This may serve as a useful test bed for developing nanostructured photonic systems.
Although emerging evidence indicates that deep-sea water contains an untapped reservoir of high metabolic and genetic diversity, this realm has not been studied well compared with surface sea water. The study provided the first integrated meta-genomic and -transcriptomic analysis of the microbial communities in deep-sea water of North Pacific Ocean. DNA/RNA amplifications and simultaneous metagenomic and metatranscriptomic analyses were employed to discover information concerning deep-sea microbial communities from four different deep-sea sites ranging from the mesopelagic to pelagic ocean. Within the prokaryotic community, bacteria is absolutely dominant (~90%) over archaea in both metagenomic and metatranscriptomic data pools. The emergence of archaeal phyla Crenarchaeota, Euryarchaeota, Thaumarchaeota, bacterial phyla Actinobacteria, Firmicutes, sub-phyla Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria, and the decrease of bacterial phyla Bacteroidetes and Alphaproteobacteria are the main composition changes of prokaryotic communities in the deep-sea water, when compared with the reference Global Ocean Sampling Expedition (GOS) surface water. Photosynthetic Cyanobacteria exist in all four metagenomic libraries and two metatranscriptomic libraries. In Eukaryota community, decreased abundance of fungi and algae in deep sea was observed. RNA/DNA ratio was employed as an index to show metabolic activity strength of microbes in deep sea. Functional analysis indicated that deep-sea microbes are leading a defensive lifestyle.
Background: The use of culture-independent nucleic acid techniques, such as ribosomal RNA gene cloning library analysis, has unveiled the tremendous microbial diversity that exists in natural environments. In sharp contrast to this great achievement is the current difficulty in cultivating the majority of bacterial species or phylotypes revealed by molecular approaches. Although recent new technologies such as metagenomics and metatranscriptomics can provide more functionality information about the microbial communities, it is still important to develop the capacity to isolate and cultivate individual microbial species or strains in order to gain a better understanding of microbial physiology and to apply isolates for various biotechnological applications.
Results: We have developed a new system to cultivate bacteria in an array of droplets. The key component of the system is the microbe observation and cultivation array (MOCA), which consists of a Petri dish that contains an array of droplets as cultivation chambers. MOCA exploits the dominance of surface tension in small amounts of liquid to spontaneously trap cells in well-defined droplets on hydrophilic patterns. During cultivation, the growth of the bacterial cells across the droplet array can be monitored using an automated microscope, which can produce a real-time record of the growth. When bacterial cells grow to a visible microcolony level in the system, they can be transferred using a micropipette for further cultivation or analysis.
Conclusions: MOCA is a flexible system that is easy to set up, and provides the sensitivity to monitor growth of single bacterial cells. It is a cost-efficient technical platform for bioassay screening and for cultivation and isolation of bacteria from natural environments.
The unicellular microalga Haematococcus pluvialis has emerged as a promising biomass feedstock for the ketocarotenoid astaxanthin and neutral lipid triacylglycerol. Motile flagellates, resting palmella cells, and cysts are the major life cycle stages of H. pluvialis. Fast-growing motile cells are usually used to induce astaxanthin and triacylglycerol biosynthesis under stress conditions (high light or nutrient starvation); however, productivity of biomass and bioproducts are compromised due to the susceptibility of motile cells to stress. This study revealed that the Photosystem II (PSII) reaction center D1 protein, the manganese-stabilizing protein PsbO, and several major membrane glycerolipids (particularly for chloroplast membrane lipids monogalactosyldiacylglycerol and phosphatidylglycerol), decreased dramatically in motile cells under high light (HL). In contrast, palmella cells, which are transformed from motile cells after an extended period of time under favorable growth conditions, have developed multiple protective mechanisms - including reduction in chloroplast membrane lipids content, downplay of linear photosynthetic electron transport, and activating nonphotochemical quenching mechanisms - while accumulating triacylglycerol. Consequently, the membrane lipids and PSII proteins (D1 and PsbO) remained relatively stable in palmella cells subjected to HL. Introducing palmella instead of motile cells to stress conditions may greatly increase astaxanthin and lipid production in H. pluvialis culture.