Streptavidin - A Versatile Binding Protein for Solid-Phase Immunoassays

Streptavidin, a tetrameric protein secreted by Streptomyces avidinii, binds tightly to a small growth factor biotin. One of the numerous applications of this high-affinity system comprises the streptavidin-coated surfaces of bioanalytical assays which serve as universal binders for straightforward immobilization of any biotinylated molecule. Proteins can be immobilized with a lower risk of denaturation using streptavidin-biotin technology in contrast to direct passive adsorption. The purpose of this study was to characterize the properties and effects of streptavidin-coated binding surfaces on the performance of solid-phase immunoassays and to investigate the contributions of surface modifications. Various characterization tools and methods established in the study enabled the convenient monitoring and binding capacity determination of streptavidin-coated surfaces. The schematic modeling of the monolayer surface and the quantification of adsorbed streptavidin disclosed the possibilities and the limits of passive adsorption. The defined yield of 250 ng/cm² represented approximately 65 % coverage compared with a modelled complete monolayer, which is consistent with theoretical surface models. Modifications such as polymerization and chemical activation of streptavidin resulted in a close to 10-fold increase in the biotin-binding densities of the surface compared with the regular streptavidin coating. In addition, the stability of the surface against leaching was improved by chemical modification. The increased binding densities and capacities enabled wider high-end dynamic ranges in the solid-phase immunoassays, especially when using the fragments of the capture antibodies instead of intact antibodies for the binding of the antigen. The binding capacity of the streptavidin surface was not, by definition, predictive of the low-end performance of the immunoassays nor the assay sensitivity. Other features such as non-specific binding, variation and leaching turned out to be more relevant. The immunoassays that use a direct surface readout measurement of time-resolved fluorescence from a washed surface are dependent on the density of the labeled antibodies in a defined area on the surface. The binding surface was condensed into a spot by coating streptavidin in liquid droplets into special microtiter wells holding a small circular indentation at the bottom. The condensed binding area enabled a denser packing of the labeled antibodies on the surface. This resulted in a 5 - 6-fold increase in the signal-to-background ratios and an equivalent improvement in the detection limits of the solid-phase immunoassays. This work proved that the properties of the streptavidin-coated surfaces can be modified and that the defined properties of the streptavidin-based immunocapture surfaces contribute to the performance of heterogeneous immunoassays.

Unique features of odorant-binding proteins of the parasitoid wasp Nasonia vitripennis revealed by genome annotation and comparative analyses

Insects are the most diverse group of animals on the planet, comprising over 90% of all metazoan life forms, and have adapted to a wide diversity of ecosystems in nearly all environments. They have evolved highly sensitive chemical senses that are central to their interaction with their environment and to communication between individuals. Understanding the molecular bases of insect olfaction is therefore of great importance from both a basic and applied perspective. Odorant binding proteins (OBPs) are some of most abundant proteins found in insect olfactory organs, where they are the first component of the olfactory transduction cascade, carrying odorant molecules to the olfactory receptors. We carried out a search for OBPs in the genome of the parasitoid wasp Nasonia vitripennis and identified 90 sequences encoding putative OBPs. This is the largest OBP family so far reported in insects. We report unique features of the N. vitripennis OBPs, including the presence and evolutionary origin of a new subfamily of double-domain OBPs (consisting of two concatenated OBP domains), the loss of conserved cysteine residues and the expression of pseudogenes. This study also demonstrates the extremely dynamic evolution of the insect OBP family: (i) the number of different OBPs can vary greatly between species; (ii) the sequences are highly diverse, sometimes as a result of positive selection pressure with even the canonical cysteines being lost; (iii) new lineage specific domain arrangements can arise, such as the double domain OBP subfamily of wasps and mosquitoes.

Regulatable and modulable background expression control in prokaryotic synthetic circuits by auxiliary repressor binding sites.

Expression control in synthetic genetic circuitry, for example, for construction of sensitive biosensors, is hampered by the lack of DNA parts that maintain ultralow background yet achieve high output upon signal integration by the cells. Here, we demonstrate how placement of auxiliary transcription factor binding sites within a regulatable promoter context can yield an important gain in signal-to-noise output ratios from prokaryotic biosensor circuits. As a proof of principle, we use the arsenite-responsive ArsR repressor protein from Escherichia coli and its cognate operator. Additional ArsR operators placed downstream of its target promoter can act as a transcription roadblock in a distance-dependent manner and reduce background expression of downstream-placed reporter genes. We show that the transcription roadblock functions both in cognate and heterologous promoter contexts. Secondary ArsR operators placed upstream of their promoter can also improve signal-to-noise output while maintaining effector dependency. Importantly, background control can be released through the addition of micromolar concentrations of arsenite. The ArsR-operator system thus provides a flexible system for additional gene expression control, which, given the extreme sensitivity to micrograms per liter effector concentrations, could be applicable in more general contexts.

Functional impact of genetic variation on gene expression

Numerous links between genetic variants and phenotypes are known and genome-wide association studies dramatically increased the number of genetic variants associated with traits during the last decade. However, how changes in the DNA perturb the molecular mechanisms and impact on the phenotype of an organism remains elusive. Studies suggest that many traitassociated variants are in the non-coding region of the genome and probably act through regulation of gene expression. During my thesis I investigated how genetic variants affect gene expression through gene regulatory mechanisms. The first chapter was a collaborative project with a pharmaceutical company, where we investigated genome-wide copy number variation (CNVs) among Cynomolgus monkeys (Macaca fascicularis) used in pharmaceutical studies, and associated them to changes in gene expression. We found substantial copy number variation and identified CNVs linked to tissue-specific expression changes of proximal genes. The second and third chapters focus on genetic variation in humans and its effects on gene regulatory mechanisms and gene expression. The second chapter studies two human trios, where the allelic effects of genetic variation on genome-wide gene expression, protein-DNA binding and chromatin modifications were investigated. We found abundant allele specific activity across all measured molecular phenotypes and show extended coordinated behavior among them. In the third chapter, we investigated the impact of genetic variation on these phenotypes in 47 unrelated individuals. We found that chromatin phenotypes are organized into local variable modules, often linked to genetic variation and gene expression. Our results suggest that chromatin variation emerges as a result of perturbations of cis-regulatory elements by genetic variants, leading to gene expression changes. The work of this thesis provides novel insights into how genetic variation impacts gene expression by perturbing regulatory mechanisms. -- De nombreux liens entre variations génétiques et phénotypes sont connus. Les études d'association pangénomique ont considérablement permis d'augmenter le nombre de variations génétiques associées à des phénotypes au cours de la dernière décennie. Cependant, comprendre comment ces changements perturbent les mécanismes moléculaires et affectent le phénotype d'un organisme nous échappe encore. Des études suggèrent que de nombreuses variations, associées à des phénotypes, sont situées dans les régions non codantes du génome et sont susceptibles d'agir en modifiant la régulation d'expression des gènes. Au cours de ma thèse, j'ai étudié comment les variations génétiques affectent les niveaux d'expression des gènes en perturbant les mécanismes de régulation de leur expression. Le travail présenté dans le premier chapitre est un projet en collaboration avec une société pharmaceutique. Nous avons étudié les variations en nombre de copies (CNV) présentes chez le macaque crabier (Macaca fascicularis) qui est utilisé dans les études pharmaceutiques, et nous les avons associées avec des changements d'expression des gènes. Nous avons découvert qu'il existe une variabilité substantielle du nombre de copies et nous avons identifié des CNVs liées aux changements d'expression des gènes situés dans leur voisinage. Ces associations sont présentes ou absentes de manière spécifique dans certains tissus. Les deuxième et troisième chapitres se concentrent sur les variations génétiques dans les populations humaines et leurs effets sur les mécanismes de régulation des gènes et leur expression. Le premier se penche sur deux trios humains, père, mère, enfant, au sein duquel nous avons étudié les effets alléliques des variations génétiques sur l'expression des gènes, les liaisons protéine-ADN et les modifications de la chromatine. Nous avons découvert que l'activité spécifique des allèles est abondante abonde dans tous ces phénotypes moléculaires et nous avons démontré que ces derniers ont un comportement coordonné entre eux. Dans le second, nous avons examiné l'impact des variations génétiques de ces phénotypes moléculaires chez 47 individus, sans lien de parenté. Nous avons observé que les phénotypes de la chromatine sont organisés en modules locaux, qui sont liés aux variations génétiques et à l'expression des gènes. Nos résultats suggèrent que la variabilité de la chromatine est due à des variations génétiques qui perturbent des éléments cis-régulateurs, et peut conduire à des changements dans l'expression des gènes. Le travail présenté dans cette thèse fournit de nouvelles pistes pour comprendre l'impact des différentes variations génétiques sur l'expression des gènes à travers les mécanismes de régulation.

Genetically-defined deficiency of mannose-binding lectin is associated with protection after experimental stroke in mice and outcome in human stroke

The complement system is a major effector of innate immunity that has been involved in stroke brain damage. Complement activation occurs through the classical, alternative and lectin pathways. The latter is initiated by mannose-binding lectin (MBL) and MBL-associated serine proteases (MASPs). Here we investigated whether the lectin pathway contributes to stroke outcome in mice and humans.

Highly functional binding surface for miniaturised solid-phase immunoassays. - A spot story -

Several bioaffinity assays are based on the detection of an analyte which is bound on a solid substrate via biochemical interaction. These so called solid phase assays are based on the adhesion of the primary binding partner on a solid surface, which then binds the analyte to be detected. In this thesis work a novel solid phase based assay technology, known as spot technology, was developed. The spot technology is based on combination of high-capacity solid phases, concentrated in a spot format, utilising modified streptavidin molecules and recombinant antibody fragments. The reduction of the solid phase binding surface to a size of a spot enabled denser binding of the target molecules, providing improved signal intensities and signal-to-background ratio when applied in different solid phase immunoassays. Streptavidin-biotin interactions are commonly utilised in numerous different bioaffinity assays and the ultimate nature of streptavidin to bind biotin is among the strongest non-covalent interaction reported between two biomolecules. In this study native core streptavidin was chemically modified to provide polymerised streptavidin molecules with altered adsorption properties. These streptavidin conjugates, when coated onto polystyrene surface, provided enhanced biotin binding capacity and surface stability when compared to a reference coating constructed with native streptavidin. Furthermore, the combination of chemically modified streptavidin, sitespecifically biotinylated antibody fragments and the spot coating technology provided highly dense solid phase coating with improved binding properties. The performance of the spot assay technology was further demonstrated in different immunoassay configurations. Human thyroid stimulating hormone (TSH) and human cardiac troponin I (cTnI) were used as model analytes to show the applicability of the highly sensitive spot-based solid-phase immunoassay for detection of very low levels of analytes. It was demonstrated that the spot technology provided an assay concept with enhanced sensitivity and short turn-around times, characteristics that are highly suitable for point-of-care applications.

Building complexity in O2-binding copper complexes : site-selective metalation and intermolecular O2-binding at dicopper and heterometallic complexes derived from an unsymmetric ligand

A novel unsymmetric dinucleating ligand (LN3N4) combining a tridentate and a tetradentate binding sites linked through a m-xylyl spacer was synthesized as ligand scaffold for preparing homo- and dimetallic complexes, where the two metal ions are bound in two different coordination environments. Site-selective binding of different metal ions is demonstrated. LN3N4 is able to discriminate between CuI and a complementary metal (M′ = CuI, ZnII, FeII, CuII, or GaIII) so that pure heterodimetallic complexes with a general formula [CuIM′(LN3N4)]n+ are synthesized. Reaction of the dicopper(I) complex [CuI 2(LN3N4)]2+ with O2 leads to the formation of two different copper-dioxygen (Cu2O2) intermolecular species (O and TP) between two copper atoms located in the same site from different complex molecules. Taking advantage of this feature, reaction of the heterodimetallic complexes [CuM′(LN3N4)]n+ with O2 at low temperature is used as a tool to determine the final position of the CuI center in the system because only one of the two Cu2O2 species is formed

Synthesis and antitubercular activity of isoniazid condensed with carbohydrate derivatives

A series of 13 compounds analogous of isoniazid condensed with carbohydrate was synthesized and evaluated for their in vitro antibacterial activity against Mycobacterium tuberculosis H37Rv using Alamar Blue susceptibility test and the activity expressed as the minimum inhibitory concentration (MIC90) in μg/mL. Several compounds exhibited antitubercular activity (0.31-3.12 μg/mL) when compared with first line drugs such as isoniazid (INH) and rifampicin (RIP) and could be a good starting point to develop new compounds against tuberculosis.

ANION-BINDING AND SENSING PROPERTIES OF NOVEL RECEPTORS BASED ON N-(INDOL-3-YLGLYOXYLYL)BENZYLAMINE

Indole-based receptors such as biindole, carbazole, and indolocarbazole are regarded as some of the most favorable anion receptors in molecular recognition. This is because indole groups possess N–H groups as hydrogen-bonding donors. The introduction of amide groups in the indole framework can induce strong binding properties and good water solubility. In this study, we designed and synthesized a series of N-(indol-3-ylglyoxylyl)benzylamine derivatives as novel and simple anion receptors. The receptors derived by aryl and aliphatic amines can selectively recognize F– based on a color change from colorless-to-yellow in DMSO. The receptors derived by hydrazine hydrate can recognize F–, AcO–, and H2PO4– by similar color changes in DMSO and can even enable the selective recognition of F– in a DMSO–H2O binary solution by the naked eye. Spectrographic data indicate that complexes are formed between receptors and anions through multiple hydrogen-bonding interactions in dual solutions.

Sugarcane yellow leaf virus infection leads to alterations in photosynthetic efficiency and carbohydrate accumulation in sugarcane leaves

Infection by Sugarcane yellow leaf virus (ScYLV) causes severe leaf symptoms in sugarcane (Saccharum spp.) hybrids, which indicate alterations in its photosynthetic apparatus. To gain an overview of the physiological status of infected plants, we evaluated chlorophyll a fluorescence and gas exchange assays, correlating the results with leaf metabolic surveys, i.e., photosynthetic pigments and carbohydrate contents. When compared to healthy plants, infected plants showed a reduction in potential quantum efficiency for photochemistry of photosystem (PSII) and alterations in the filling up of the plastoquinone (PQ) pool. They also showed reduction in the CO2 net exchange rates, probably as a consequence of impaired quantum yield. In addition, reductions were found in the contents of photosynthetic leaf pigments and in the ratio chlorophyll a/chlorophyll b (chla/chlb). Carbohydrate content in the leaves was increased as a secondary effect of the ScYLV infection. This article discusses the relation of virus replication and host defense responses with general alterations in the photosynthetic apparatus and in the metabolism of infected plants.

Effect of magnesium addition on the vacancy-Mg binding free enthalpy in Al-Cr alloys

The Mg-vacancy binding free enthalpy of Al-Cr solid solution alloys with Mg addition was calculated by electrical resistivity measurements. The obtained value is lower than that obtained for dilute Al-Mg alloys with almost the same Mg content and may be attributed to the diffusion of Mg.