Dissertations / Theses on the topic 'DNA aptamers'

Le travail réalisé au cours de cette thèse a porté sur le développement de biocapteurs électrochimiques d’affinité, sensibles et sélectifs, pour la détection de l’ochratoxine A (OTA) et l’aflatoxine M1 (AFM1). Les biocapteurs développés reposent sur l’association de différents nanomatériaux pour une meilleure performance analytique. Pour construire notre transducteur, nous avons associé le polypyrrole à des dendrimères poly(amido-amine) PAMAM, ce qui a permis d’avoir de très bon rendements grâce au propriétés électriques du polypyrrole et à l’augmentation de la surface active due à la structure tridimensionnelle des dendrimères. L’utilisation d’aptamères spécifiques pour la détection des différentes mycotoxines a permis leur détection et quantification à des concentrations de l’ordre des nM, ainsi que l’élargissement des gammes dynamiques. Nous avons pu démontrer grâce à l’utilisation de dendrimères de différentes tailles que la sensibilité des biocapteurs ne provient pas uniquement de l’affinité qui existe entre les biorécepteurs et leurs molécules cibles, mais aussi des propriétés physico-chimiques du biocapteur
This aim of this work is to develop ultrasensitive electrochemical biosensors with high affinity toward ochratoxine (OTA) and aflatoxine M1 (AFM1). In order to obtain the best analytical performances, we associated nano-materials in the transducer construction: conducting polypyrrole polymer and poly(amido-amine) dendrimères. Thanks to this association, we benefited from the conducting material’s electrical properties, and the large active detection surface dendrimers. For the bimolecular sensing part, we used specific DNA aptamers which allowed us to quantify mycotoxines at nM concentrations. In addition, the different aptamer based biosensors present a very large dynamic ranges. We also demonstrated through the use of different sizes of dendrimers, that the sensitivity depend not only in the affinity between bioreceptors and their target molecules, but also in the physico-chemical properties of the biosensor

Aptamer probes for specific recognition of glioblastoma multiforme were generated using a repetitive and broad cell-SELEX-based procedure without negative selection. The 454 sequencing technology was used to monitor SELEX, and bioinformatics tools were used to identify aptamers from high throughput data. A group of aptamers were generated that can bind to target cells specifically with dissociation constants (K d ) in the nanomolar range. Selected aptamers showed high affinity to different types of glioblastoma cell lines, while showing little or no affinity to other cancer cell lines. The aptamers generated in this study have potential use in different applications, such as probes for diagnosis and devices for targeted drug delivery, as well as tools for molecular marker discovery for glioblastomas.

Aptamers are biomolecular ligands composed of nucleic acids. They can be selected to bind specifically to a range of target molecules and subsequently exploited in a fashion analogous to more traditional biomolecules such as antibodies. In this study a method for selecting new aptamers which specifically bind whole live bacterial cells is described. A non-pathogenic strain of Escherichia coli K12 was used to develop the method. A DNA library containing 100 bases long random nucleotide sequences was produced and the aptamer selection process was repeated nine times. An enzyme-linked technique was first used to detect bound aptamers thereafter fluorimetry and fluorescence microscopy methods were used for the detection. The aptamers were cloned and sequenced and the cloned aptamers produced with fluorescent labels. The E. coli K12-binding aptamers were used to demonstrate the detection of the bacterial cells in a complex food matrix, namely probiotic yogurt, by using fluorescence based detection method. The aptamer selection method with some modifications was also used to select aptamers with specificity for the food pathogens E. coli O157, Listeria monocytogenes, L. innocua, S. typhimurium and S. enteritidis. The aptamers against E. coli O157 and S. typhimurium were cloned and the sequences and the binding properties of these aptamers were analysed. The use of E. coli K12 as a target organism and the aptamer sequences presented in this study, have not previously been published in scientific literature. This is also the first report where the aptamers have been used in detection of live bacterial cells in yogurt.

Rapid Diagnostic Tests (RDTs) for malaria are restricted to a few biomarkers and antibody-mediated detection. However, the expression of commonly used biomarkers varies geographically and the sensibility of immunodetection can be affected by batch-to-batch differences or limited thermal stability. In this study we aimed to overcome these limitations by identifying a potential biomarker and by developing molecular sensors based on aptamer technology. Using gene expression databases, ribosome profiling analysis, and structural modeling, we find that the High Mobility Group Box 1 protein (HMGB1) of Plasmodium falciparum is highly expressed, structurally stable, and present along all blood-stages of P. falciparum infection. To develop biosensors, we used in vitro evolution techniques to produce DNA aptamers for the recombinantly expressed HMG-box, the conserved domain of HMGB1. An evolutionary approach for evaluating the dynamics of aptamer populations suggested three predominant aptamer motifs. Representatives of the aptamer families were tested for binding parameters to the HMG-box domain using microscale thermophoresis and rapid kinetics. Dissociation constants of the aptamers varied over two orders of magnitude between nano- and micromolar ranges while the aptamer-HMG-box interaction occurred in a few seconds. The specificity of aptamer binding to the HMG-box of P. falciparum compared to its human homolog depended on pH conditions. Altogether, our study proposes HMGB1 as a candidate biomarker and a set of sensing aptamers that can be further developed into rapid diagnostic tests for P. falciparum detection.
Grand Challenges Canada
Revisión por pares

La détection de petites molécules contribue au développement de nombreux domaines tels que la sécurité alimentaire, la sécurité intérieure, le diagnostic, le contrôle de l'environnement, etc. Cependant, la petite taille de ces cibles et leur faible concentration rendent difficile leur détection. Pour pallier à cela, des biocapteurs avec des sondes appropriées et des stratégies d'amplification du signal sont nécessaires. Parmi les éléments de reconnaissance couramment utilisés, les aptamères présentent l'avantage d'une synthèse aisée et de grandes possibilités de modification, ainsi qu'une dénaturation réversible à haute température et une tolérance élevée à la concentration en sel et au pH dans le milieu de travail. Plus important encore, la petite taille des aptamères en fait un choix idéal pour créer des structures adaptées pour la détection de petites cibles. La possibilité de couper la séquence de l'aptamère a fourni d'autres approches d’amplification de signal. Il existe deux catégories de méthodes de détection basées sur des aptamères : analyse hétérogène lorsque l'aptamère est immobilisé sur la surface ou analyse homogène lorsque le test est réalisé en solution. Nous proposons dans cette thèse une approche appliquable aux deux stratégies. L'adénosine a été utilisée comme une cible modèle pour cette preuve de concept. La détection de l'adénosine a été obtenue en combinant l'auto-assemblage de dimères d'oligonucléotides avec des extrémités pendantes correspondantes à l'aptamère coupé. Nous avons construit des structures auto-assemblées d'ADN (de 1D à 3D) avec l'adénosine comme déclencheur d'un changement structurel. La première méthode décrite dans ce travail consiste à utiliser de telles structures d'ADN combinées à l'imagerie par Résonance de Plasmons de Surface (SPRi). La SPRi est une méthode sensible à la variation d'indice optique produite par l'interaction entre les sondes immobilisées sur le prisme de l'or et la cible dans la solution. En présence d'adénosine, la structure d'ADN s'auto-assemble sur la surface de l'or et un signal a été créé. La limite de détection de l'adénosine atteinte par cette méthode est de 10 μM. La deuxième homogène méthode consiste à analyser les variations d'absorbance UV de la solution contenant les structures d'ADN puisque l'absorbance UV de l'ADN monocaténaire et du duplex ADN hybride est différente. En raison de cet effet, la température de fusion des brins d'ADN peut être déterminée par la dérivée de l'absorbance UV mesurée. Les structures d'ADN combinant les extrémités pendantes de l'aptamère coupé couplés à des oligonucléotides complémentaires présentent deux températures de fusion caractéristique de la dissociation de chaque partie. L'une correspond à l'oligonucléotide hybridé et l'autre à l'aptamère coupé liant l'adénosine. En présence d'adénosine dans la solution, la stabilité de la structure augmente et le pic de fusion de l'aptamère coupé est décalé à une température plus élevée tandis que le second pic de fusion reste identique et peut servir de référence interne. La limite de détection atteinte pour cette méthode est de 1 μM. Les structures d'ADN que nous avons proposées s'auto-assemblent de manière linéaire ou bi- ou tri-dimensionnelle : la structure 1D est une chaîne d'ADN formée par un enchainement de dimères connectés par des extrémités formées de l'aptamère scindé; La structure en 2D est une structure en forme de Y formée par un ADN simple brin avec une extrémité aptamère scindé sur chaque branche du "Y"; La structure 3D est un tétraèdre formé par quatre simple brins d'ADN avec des extrémités aptamère scindé sur les quatre sommets. En présence d'adénosine, les structures 2D et 3D peuvent s'auto-assembler et ainsi former un réseau avec les extrémités pendantes. La structure 1D a été mûrement développée pour les deux méthodes, les structures 2D et 3D ont été prouvées efficaces pour la détection, mais nécessitent encore plus d'efforts pour permettre une détection optimisée
The detection of small molecules contributes to the development of many fields such as food safety, homeland security, diagnose, environment control, etc. However, their small size and low concentration are the usual cause of limitations in their detection. In order to improve the detection, biosensors with appropriate probes and signal amplification strategies are required. Amongst the commonly used recognition elements, aptamer has the advantage of easier mass production and modification, reversible denaturation at high temperature and high tolerance of salt concentration and pH in the working environment. More importantly its small size made it an ideal choice for creating delicate structures for the detection of small targets. The possibility of splitting the aptamer sequence has provided more approaches for amplification purpose. There are two categories of detecting methods based on aptamers: heterogeneous analyzation where the aptamer is immobilized on a surface or homogeneous analyzation where the assay is performed in solution. In this thesis, we proposed an amplification method useful for both heterogeneous and homogeneous assays. Adenosine was used as a proof of concept target. The detection of Adenosine was achieved by combining the self-assembly of oligonucleotide dimers with split-aptamer dangling ends. We constructed self-assembled DNA structures (from 1D to 3D) with Adenosine as the trigger for a structural change. The heterogeneous assay is based on in Surface Plasmon Resonance imaging (SPRi). SPRi is a method sensitive to the change of refraction index created by the interaction between the probes immobilized on the gold surface and the targets in the flowing solution. With the presence of Adenosine in the solution, the DNA structure is self-assembled on the gold surface and the signal was created. The detection limit achieved by this method was 10 µM. The second homogeneous assay is based on the melting profile of the solution determined from the absorbance of UV light (260 nm wavelength). The UV absorbance of single strand DNA and hybridized DNA duplex is different. Due to this effect, the melting temperature could be obtained from the UV absorbance measured. The DNA structures combining self-complementary oligonucleotides and split-aptamer dangling ends have two melting temperatures, one correspond to the oligonucleotides and the other to the split-aptamer. In presence of Adenosine in the solution the strength in the binding is increased. As a result, the melting peak of the split-aptamer shifted to higher temperature while the second melting peak correspond the oligonucleotide remains the same as an internal reference. The detection limit achieved for this method was 1 µM. The DNA structures we proposed varied from 1D to 3D: the 1D structure was a DNA chain formed by a series of dimers connected through split-aptamer dangling ends; the 2D structure was a Y shape structure formed by three single-strand DNA with a split-aptamer dangling end on each branch of the “Y”; the 3D structure was a tetrahedron formed by four single-strand DNA with split-aptamer dangling ends on the four vertexes. With presence of Adenosine, 2D and 3D structures can further form a network with the dangling ends. The 1D structure has been maturely developed for the two detection methods, the 2D and 3D structures have been proven effective for detection but still require more efforts to reach perfection

Nucleic acid aptamers are a group of molecules which emerge from large random sequence pools through in vitro selection, a process called Systematic Evolution of Ligands by EXponential enrichment (SELEX). These recognition molecules are a potential alternative to antibodies and offer a greater thermal stability, robustness and chemical versatility. The first part of this thesis describes the selection of an aptamer against acetylcholinesterase (AChE) using conventional in vitro selection process. The pool enrichment was observed during 15 rounds of selection and a chosen aptamer, R15/19, was characterized. The R15/19 binds with dissociation constant of 55±8pM but does not inhibit the enzyme. This aptamer can be used as a part of immobilization mediator. However, this manual in vitro selection procedure is time consuming as it needs multiple rounds of selection. Therefore, a novel aptamer selection method has been developed that is aimed at shortening selection times. Taking an advantage of multiple interactions, it is possible to generate a single sequence that contains two binding sites through a single selection process. The approach is adopted from proximity dependent DNA ligation assay (PLA) and called 'proximity ligation selection' (PLS). Instead of using one random pool for selection, with PLS, we use two. These two starting pools were designed to contain different additional sequences, apart from the primer and random regions, at either the 5' or the 3' end. The basis of this method is only in the presence of target protein molecules, is there a close proximity of pairs of bound sequences. And only if those pairs are in an appropriate orientation in which the 3' hydroxyl terminal of one sequence is sufficiently close to the 5'-phosphate terminal of the other sequence, are the extended free ends of each member of the proximity oligonucleotide pairs were brought closely by base-pairing to subsequently added connector oligonucleotide. The hybridized complexes are then joined using a DNA ligase, allowing the formation of a ligated product that contains a dual binding site on the target. Thrombin and lysozyme were used as model targets. For thrombin, none of the PLS products showed a G-quadruplex signature in CD spectroscopy. Using the SPR, the Kd of the aptamer dimer 2/19-5 (from F and R pool) and 4/19-7 (from TBA29plus and R pool) are 1.6μM and 1.2μM, respectively. The ELONA test was used to measure a Kd for each monomer and there was no binding observed from each monomer of the aptamer against thrombin. In the case of lysozyme, the SPR and ELONA revealed the Kd of the aptamer dimer (from two starting pools) of 8.7μM and 9nM, respectively. The Kd of each constitute of anti-lysozyme aptamer dimer using ELONA showed the Kd of 35.5 and 51.5nM. The Kd obtained from SPR was high, suggesting a low binding affinity. We assume that the aptamer molecule can freely fold into a favored structure once it meets the target and thus can perform binding better than in the SPR. Although we did not succeed in generating aptamer dimers that had higher affinity than monomers, there are several steps in the procedure that need to be further improved.

DNA-functionalized gold nanoparticles (DNA-NPs) have enormous potential as building blocks for materials due to their ability to specifically recognize and respond to target molecules and surfaces. The ability of DNA aptamers to adopt different conformations and bind either complementary DNA sequences or analyte molecules allows them to mediate nanoparticle assembly or disassembly, generating selective colorimetric responses. Aptamer-mediated nanoparticle assembly and disassembly is sensitive to the nanoparticle ligand shell composition and structure, yet these topics have not been extensively explored. In this dissertation, a method for determining the ligand shell composition of DNA-NPs is described and a framework for understanding the impact of the DNA assembly arrangement and recognition strand density upon aptamer-mediated nanoparticle assembly and disassembly is developed. Design rules for creating sensors with desired properties are elucidated, leading to creation of sensors with improved detection limits and quantification ranges. A technique was needed to determine the number of DNA strands of any base composition attached to gold nanoparticles (AuNPs) of any core size. A rapid, convenient and inexpensive method to quantify the number of label-free DNA strands attached to AuNPs was therefore developed. This technique was extended to determine two different DNA sequences bound to AuNPs using UV-visible and fluorescence spectroscopy. Based on the results of quantifying the ligand shells of DNA-NPs functionalized with two sequences, disulfide-terminated DNA non-specifically adsorbs and then rearranges to specifically bind the gold surface. The position of the AuNPs and DNA strands within DNA-NP assemblies had a profound influence on their ability to assemble and sense adenosine. Assemblies designed for large inter-AuNP spacing were stable but unable to sense adenosine. Assemblies designed for short inter-AuNP spacing were unstable until the DNA ligand shell was diluted. AuNPs functionalized with the fewest number of aptamers produced assemblies with the lowest detection limit and apparent disassociation constant and the largest analyte quantification range. Increasing the number of aptamer strands per AuNP increased the cooperativity of the AuNP disassembly response to adenosine. This dissertation includes previously unpublished co-authored material.

Detection of cancer at the early stages greatly increases the chance for successful treatment and favourable prognosis for patients. However, a liquid-based biopsy has yet to be developed for most cancers. Extracellular vesicles (EVs) are an attractive candidate for early cancer detection since their surface proteome mirrors the cell of origin. Thus, there is a need for the development of reliable probes that can detect cancer derived EVs. In this thesis, the VBS-1 aptamer was developed to selectively bind to triple-negative breast cancer cell line derived EVs. Initially, several EV isolation methods were compared and isolated EVs were validated and characterized. Aptamer clones were developed by Systematic Evolution of Ligands by Exponential Enrichment to EVs isolated by differential ultracentrifugation and their binding was validated by flow cytometry. The binding partner of the selected VBS-1 aptamer was identified by LC-MS/MS to be the transmembrane protein ATP1A1. The presence of an ATP1A1-positive EV population was validated by flow cytometry. The selected aptamer may find further application in biosensors for the detection of EVs as cancer biomarkers in biological fluids.

This dissertation presents the development of an alternative aptamer screening process, Competition-Induced Selection of Ligands (CISL), and its use in screening for ssDNA aptamers for gold substrates. Gold substrates are presented as the nonnucleotide target for implementing CISL as a novel aptamer screening approach. Chapter 1 provides an overview of the in vitro selection of oligonucleotide aptamers, the polymerase chain reaction that is a key step in the aptamer screening process, the synthesis and properties of gold nanoparticles and the biomolecule-mediated formation of inorganic nanoparticles. Chapter 2 presents the goals and objectives of this thesis along with an organizational overview of the dissertation. Chapter 3 describes the experimental techniques and optimizations pertinent to the development of the CISL aptamer screening process. Chapter 4 investigates the effects of various nucleic acid additions during the seed-mediated growth of gold nanoparticles. Chapter 5 discusses the use of CISL in screening for ssDNA aptamer candidates for spherical gold nanoparticles (AuNPs) and the primary and secondary structure analysis of identified sequences. Chapter 6 presents the use of CISL in screening for ssDNA aptamer candidates for planar gold substrates (PlanarAu) and also includes primary and secondary structure analysis of identified sequences accompanied with an incubation study to provide a “frequency” ranking of aptamers as adsorbate species on PlanarAu. Chapter 7 offers concluding remarks and ideas for future expansion and applications of this work.

>Magister Scientiae - MSc
Tuberculosis (TB) is a global health problem and rated as the second leading cause of death after HIV/AIDS. Transmission of TB from one person to the next is very rapid in crowded communities. Therefore, it is crucial to identify people who are infected as quickly as possible not only to provide treatment but also to prevent the spread of the disease. Current TB diagnostic tests such as the culture and sputum smear tests are time-consuming, while rapid tests make use of antibodies that are costly and have low sensitivity and stability. Great improvement has been observed when aptamers are used in place of antibodies in rapid diagnostic tests such as lateral flow devices (LFDs). Therefore, the current study aims to synthesize and identify aptamers against serum biomarkers for development of rapid TB diagnostic tests such as a lateral flow assay. Several TB serum biomarkers have been identified and can be used for the diagnosis of TB. TB biomarkers expressed in serum samples were identified through in silico approach. The biomarkers were expressed in bacterial systems using recombinant DNA technology. The recombinant proteins were purified by affinity chromatography and further used as targets for the selection of aptamers using Systemic Evolution of Ligands by EXponential enrichment (SELEX). Aptamers for the selected biomarkers were synthesized based on magnetic-bead based SELEX and characterized by electrophoretic mobility shift assay (EMSA), Surface Plasmon resonance (SPR) and MicroScale Thermophoresis (MST). Six putative TB serum biomarker proteins were selected from literature, namely, Insulin-like Growth Factor Binding Protein 6 (IGFBP6), Interferon-stimulated Gene 15 (ISG15), Calcium Binding Protein (S100A9), Retinol Binding Protein 4 (RBP4), Granzyme A (GrA), and Transgelin-2 (TAGLN2). The biomarkers were recombinantly expressed and purified after which they were used as targets in SELEX for aptamers synthesis. Aptamers were analysed by in silico method and the ones with highly conserved motifs were selected. The selected aptamers were synthesized and later characterized. The aptamers that show high affinity and specificity for the biomarkers will be used for the fabrication of a rapid lateral flow device for TB screening. Such a test would allow for a short diagnostic turnaround time, and hence expedite treatment.

Deoxyribonucleic acid (DNA) aptamers are oligonucleotides with high specificity and affinity for non-nucleotide targets ranging from molecular species to cellular proteins. Their high affinity, rapid synthesis, and the ease with which they can be chemically modified to include convenient chemical groups (e.g. amine group on 5’ end) make them excellent adaptable ligands for use in colloidal drug delivery vehicles for both uptake and release of therapeutic agents. This work uses pre-identified aptamers for vascular endothelial growth factor (VEGF) to investigate the design of one such vehicle for controlled uptake and release of target therapeutics and analyzes the ability of particle-immobilized aptamers to bind both nucleotide and non-nucleotide targets. Aptamer sequences are immobilized on colloidal microspheres and binding activity of both the primary DNA and protein targets are directly monitored using flow cytometry. Additionally, the dual nature of aptamer-target binding is further investigated by evaluating the effects of simultaneous and serial incubation of the primary targets. Finally, the ability to recover the functionality of the aptamer is evaluated after displacement of the primary DNA target through DNA mediated interactions. It has been shown that the nature of aptamer-target interactions are complex in nature, requiring optimization for each species incorporated into a delivery vehicle; however, partial recovery of aptamer functionality was achieved after hybridization with the primary DNA target.

Biological reagents that recognize target molecules with high affinity and specificity are widely used as capture agents, diagnostic reagents, and therapeutics. Through their ability to adopt structures that confer binding affinity for a target, aptamers represent one major class of such reagents. However, their use is limited by the general inability of current selection methods to reliably discover high-quality aptamers. Inefficiencies in their selection are due in part to a lack of fundamental understanding of the mechanisms underpinning each step in the screening process. This thesis reports on a series of studies conducted to define the factors and mechanisms currently limiting aptamer selections. That knowledge is then used to create highly effective strategies and technologies for ameliorating each limitation affecting their selection. The resulting collection of improvements is integrated into a novel selection workflow termed “Hi-Fi SELEX”. Those improvements include i) application of a novel “competent library” that eliminates fixed-region interference effects during selection, ii) development of effective chemistries to optimally retain desirable library members, iii) invention of simple methods to accurately quantify retained library diversity and mean binding affinity after each selection round, and iv) development of emulsion PCR methods to eliminate generation of amplification artifacts and v) achieve stoichiometric recovery of the desired single-stranded aptamer library. The resulting discovery platform greatly improves the reliability and speed in which useful panels of lead aptamers against several clinically-relevant targets are discovered. Following initial selection of candidate aptamers based on binding affinity, further screening is typically required, in part to ensure target-specific binding – a performance need shared by antibodies selected against specific targets. However, moderate to high-throughput methods to efficiently screen panels of candidates for binding specificity are lacking. A new technology enabling label-free specificity screening of antibody or aptamer populations at suitable throughputs was therefore established at the proof-of-concept level. The novel microfluidic SPRi arrays described permit multiplexed detection of lead candidates by quantifying both equilibrium binding constants and binding kinetics for each interaction in an element-addressable fashion. The technology offers the ability to independently interrogate candidate affinity reagents and then recover those samples for downstream analysis.
Applied Science, Faculty of
Graduate

Antimicrobial resistance presents a serious, worldwide threat to public health. New tools are required to combat the evolving problem. Here we report the systematic evolution of nucleic acid ligands to live Escherichia coli cells. Two SELEX methods were used to generate aptamers to the HB101: pAT153 strain of antibiotic resistant bacterial cells. These different Cell-SELEX methods were compared to analyse how DNA adapts to selective evolutionary pressure. The first method was performed using asymmetric PCR as the mechanism of single strand regeneration, with target cells taken from bacterial colonies. The second method used a novel strand protection exonuclease method for single strand regeneration and target cells taken from live cultures. Sequences evolved during both methods were analysed and stored in plasmid libraries. These methods produced a combined total of fifty-eight putative DNA ligands (aptamers). These sequences were analysed using alignment and structural prediction software and six candidate molecules were taken for further analysis. Evolved sequences were synthesised and target cell binding assays were performed using fluorescence laser-scanning confocal microscopy to visualise binding. A number of control sequences were also analysed. These included scrambled versions of each aptamer sequence, complementary oligonucleotides to deform binding structures and positive control sequences of published cell binding aptamers. Five of the six novel sequences exhibited cell specific localisation. Two of these sequences, named Oligo 3.1 and Oligo 4.4 (one from each SELEX Method) demonstrated apparent ligand binding affinities in the low-micromolar range (KD app Oligo 3.1= 3.35 μM. KD app Oligo 4.4 = 6.69 μM).

Alterations in the expression of vital proteins of the cellular signalling pathways are at the forefront of molecular abnormalities established in cancer. Therefore, to understand the fundamental mechanisms of cancer initiation and progression, characterisation of tumour markers, such as key proteins like MUCIN that are highly expressed in malignant cells, is crucially important. Rapid drug discovery is a pressing need in the pharmaceutical industry. Agents that are able to bind tightly and selectively to the surface of diseased cells would greatly benefit both disease diagnosis and treatment. An aptamer is a functional oligonucleotide that has the appropriate sequence and structure to form a complex, possessing great affinity and selectivity for its target. Aptamers of high affinity and specificity have been successfully generated against the MUCIN splice variant from a non-conventional single round SELEX system termed SimpLex. The 72 base long aptamers were truncated to an ideal therapeutic length of 25 bases long via two different means. The first entails eliminating the primers and leaving only the central variable region and the second involves truncation of the aptamers based on their predicted secondary structure. Thu~, three aptamer versions were taken for further investigation, the complete and two truncated ones. However, as is it often the case, aptamers lack in vivo stability and are prone to rapid renal filtration and therefore require modification to achieve their full potential in vivo as therapeutic agents. Thus, the MUCIN selected aptamers were conjugated with a 32 kDa polyethylene glycol (pEG) polymer to increase their molecular weight to ea. 40 kDa, an appropriate size to avoid rapid renal clearance. The truncated aptamers, the pegylated aptamers and the full length aptamers were characterised employing qualitative techniques which, include EMSA, affinity column chromatography and DNA thermal denaturation studies. The aptamers were further assessed for their binding, specificity and internalisation by F ACs and fluorescence microscopy. The characterisation studies have shown that the anti-Ml.Cl/Y aptamers possess affinity and specificity for the MUCIfY protein and the conjugation of the aptamers to PEG has further enhanced their binding and internalisation potential. These encouraging results display potential future prospects as therapeutic agents for the pegylated aptamers and warrants their further future evaluation in in vivo studies.

Background. Inclusion Body Myositis (IBM) is the most commonly diagnosed inflammatory muscle disease associated with aging. It’s characterised by degeneration of skeletal muscles that inevitably leads to the loss of mobility. The mechanisms that drive IBM pathogenicity remain poorly understood. However, studies reveal that on average 50% of IBM sera contains circulating autoantibodies targeting cytosolic 5’-nucleotidase 1A (cN1A) – a metabolic enzyme that is highly expressed in skeletal muscles. Accumulating evidence suggests that anti-cN1A antibodies may play a role in IBM pathogenesis making them an attractive target for aptamer development. Problem. IBM remains refractive to current immunosuppressive treatments. Preventing anti-cN1A antibodies binding to their target protein may reduce the severity of IBM symptoms and halt the disease progression. Aim. Within this project, we aim to select DNA aptamers that conjugate with anti-cN1A binding sites and prevent their interaction with cN1A protein. Results. Sera of forty-eight IBM patients were screened with enzyme-linked immunosorbent assay (ELISA) against purified cN1A protein. Eighteen patients (38%) were found seropositive. 6.5 mg of polyclonal anti-cN1A were purified out of 90 ml of a selected IBM patient’s blood. Their biological activity against cN1A was confirmed by ELISA. Purified anti-cN1A were used as a target for aptamer selection. Initial library of single-stranded DNA fragments (aptamers) was synthesised with a 40-nucleotide randomised region flanked by constant primer-binding sites. Neutralising aptamers were selected through the process of systematic evolution of ligands by exponential enrichment (SELEX). Two complete cycles of selection against anti-cN1A were performed. Selected aptamers were assayed for their neutralising capacity by ELISA. Anti-cN1A were pre-incubated with the aptamers at various concentrations. Following a one-hour incubation, the ELISA-obtained absorbance remained unchanged or increased in a dose-dependent manner. While upon an extended overnight incubation, a 25% decrease in absorbance values corresponding to a 35% decrease in anti-cN1A binding was observed. Conclusions. The methodology developed during this project resulted in the generation of aptamers that conjugate with anti-cN1A binding sites. Following two SELEX cycles, the selected aptamers demonstrate 35% neutralisation capacity upon prolonged incubation with the immunoglobulins. Our results strongly suggest that the aptamers target monomeric IgG and IgA while having low or no affinity for IgM anti-cN1A.

Since the discovery was made that DNA is capable of functions beyond genetic storage and propagation, much work has been done to explore the non-canonical abilities of this molecule. Two functionalities of DNA are of particular interest. The first is the ability of DNA to act as a catalyst, and the second is the ability of the molecule to bind with high affinity to various biological and non-biological surfaces. Although numerous DNA catalysts and binders (termed “aptamers”) have been identified using the five nucleoside triphosphates found in nature, the functionality of the molecule can be expanded with the addition of protein-like functional groups to the base moiety of the nucleotide. Several modified nucleotides have been developed previously and used to discover novel catalysts and aptamers. However, the successful development of modified-DNA aptamers has thus far been limited, particularly with respect to biological targets that might be of use in a clinical or commercial context. Moreover, technical challenges in the field exist that have not yet been adequately addressed. Most significantly, the employment of the modified molecules in the discovery process can be problematic as they are not ideal substrates for many basic molecular biology procedures, notably polymerase chain reaction. New techniques to overcome these difficulties are needed and few have been developed. This thesis will focus on two aspects of modified DNA research. The first is the application of modified DNA in aptamer discovery. Starting from the hypothesis that additional functional groups will confer chemical advantages in traditionally challenging aptamer selections, the objective was to identify a modified DNA aptamer for whole bacterial cells in vitro. Several promising phenol-dUTP-modified aptamer sequences were identified using a modified SELEX procedure. The specificity and affinity of these sequences for the target bacterial strain were investigated. The second aim of this work is to address technical problems encountered when working with modified DNA in order to develop catalysts or aptamers. Specifically, a novel selection scheme was designed that eliminated the need to amplify modified DNA. Research was conducted to develop and validate this selection system as a viable route to discovery of DNA catalysts.
Science, Faculty of
Chemistry, Department of
Graduate

DNA aptamers have been studied since their inception in 1990, but have only targeted membrane and serum proteins in therapeutics. Their potential as inhibitors of protein function is hampered by their inability to efficiently enter cells in order to function. Surmounting this hurdle is worthwhile since the inhibition of protein-protein interactions is not achievable by small molecule pharmaceuticals alone. Herein we target an intracellular ubiquitin ligase WWP1, which is known to complex with Schnurri3 and polyubiquitinate Runx2, thus targeting it for proteosomal destruction. Since Runx2 is the key transcriptional regulator of osteoblast differentiation, WWP1 inhibition may encourage osteoblast differentiation, and by extension force bone deposition in osteoporosis sufferers. By targeting WWP1 we attempt to intervene in intracellular protein interactions with an aptamer for the first time. To begin this effort we cloned, expressed, and purified three functionally important truncations of WWP1. The final protein pools were highly concentrated above 2 mg/mL, approximately 95% pure, and were found to be acceptably soluble after assessment in various buffers. DNA aptamers were then selected against these WWP1 truncations using the established SELEX method while monitoring the progression of the enrichment with PCR. After 12 selection rounds of increasing stringency, pools were sequenced and assessed for homogeneity and secondary structure. Several groups of enriched and identical DNA sequences were obtained with no obvious pattern in secondary structure seen between them. While focusing on sequences specific for the active site containing C-lobe, we then evaluated the aptamers for their ability to bind key functional regions of WWP1 and inhibit its function as an enzyme. For the most potent aptamer from the C-lobe pool, an Electrophoretic Mobility Shift Assay (EMSA) estimated a Ki of around 2 μM. Furthermore, a HECT ubiquitin ligase activity assay was developed to evaluate inhibition, and an IC50 of around 100 μM was found for the most inhibitory of three C-lobe aptamers. This aptamer was then transfected into SaOS-2 osteoblastic cells so that localization could be assessed with fluorescence microscopy. Surprisingly, both the C-lobe specific aptamer and a control sequence were found to enter the cells with or without the employment of transfection reagent. Moreover, approximately 60% migrated to the nucleus and remained there over a period of days, which implies diffusion through the Nuclear Pore Complex. Taken together, this work introduces an alternative approach to disease therapy by targeting intracellular proteins with aptamers, and may have significant implications for expanding the therapeutic applications of nucleic acid aptamers in the future.
published_or_final_version
Biochemistry
Doctoral
Doctor of Philosophy

Tuberculosis is a curable disease with an average treatment success rate of 86 %. Despite this, there were an estimated 1.5 million deaths due to tuberculosis in 2013, most of which occurred in low and middle income countries. In order to overcome tuberculosis in developing countries innovation in diagnostics is key to administering treatment. While detection of whole mycobacteria has been favoured in the past to diagnose tuberculosis, culturing mycobacteria is costly and microscopy is often not sensitive enough due to low bacterial loads. Detection of Mycobacterium tuberculosis biomarkers in urine, a safe and easy specimen to test, could offer a cost effective and simple solution to identify patients with tuberculosis. Enzyme linked immunosorbent assays (ELISAs) were performed on concentrated tuberculosis patient urine to detect two M. tuberculosis biomarkers: lipoarabinomannan (LAM) and early secreted antigen-6 kDa (ESAT-6). Concentrating urine improved the detection of LAM in human immunodeficiency virus (HIV) negative patients and patients with a CD4 count > 200 cells/µl. ESAT-6 was not detected by ELISA due to a high background signal caused by the available antibodies cross reacting with a human protein present in urine which was identified by western blot and mass spectrometry. Targeted mass spectrometry did not detect ESAT-6 or its dimer partner, culture filtrate protein-10 kDa (CFP-10) in tuberculosis positive patient urine. Since concentrating urine samples is impractical in a clinical setting a more sensitive diagnostic is needed to detect LAM in urine and ESAT-6 or CFP-10 in other samples. Aptamers can be packed more densely on biosensor surfaces increasing the dynamic range of detection while matching the affinity that an antibody has for a biomarker. Chemically modified DNA aptamers were isolated for LAM and the ESAT-6.CFP-10 dimer. The aptamers were characterised by enzyme linked oligonucleotide assays (ELONAs) and biolayer interferometry. One aptamer bound with high affinity to ESAT-6 while one aptamer bound with low affinity to LAM. The use of aptamers as capture agents for detecting biomarkers in biological specimens thus appears to be a viable option for diagnosing tuberculosis, although availability and concentration of individual biomarkers seems likely to remain key to the choice of specimen in which to make diagnostic measurements.

The ATP gated cation channel P2X7, is a cell surface protein whose function and activation is linked with several human diseases. The aim of this project was to raise DNA Aptamers targeting specifically the extracellular domain of the P2X7 protein with the hope to use these new ligands as a method for the study of the biological functions of the P2X7 protein and its potential as a therapeutic target. The strategy involved isolating Aptamers that would bind to human em~ryonic kidney cells transformed with a construct which forced the cells to express native mouse P2X7 on its cell surface (POSITIVE SELECTION), whilst not binding to untransformed non-P2X7 expressing cells (NEGATIVE SELECTION). Two candidate sequences from this selection emerged as, what we called, "winners" which were the most likely sequences to putatively bind mP2X7 (work ongoing). Interestingly using the procedure in reverse, that is to create Aptamers that specifically bind to non-transformed cells and not transformed cells, also produced viable putative Aptamers, which, once labelled, showed selective binding. These results are discussed in relation to future applications and the potential insights and implications for the study of cell surface protein expression patterns. 3

Les aptamères sont des oligomères d'ADN ou d'ARN simple brin qui, en se repliant sous forme de structures tridimensionnelles peuvent avoir des interactions fortes et spécifiques envers un certain nombre de cibles. L'objectif de cette thèse a été de compléter les études existantes sur l'utilisation de l'électrophorèse capillaire (CE) et les aptamères afin de mettre au point une méthode de sélection d'aptamères par CE couplée à la fluorescence induite par laser et le séquençage haut-débit Illumina. Dans un premier temps, nous avons mis au point une méthode de détection et de séparation par électrophorèse capillaire couplée à la double détection UV-LEDIF d'une banque d'ADN en interaction avec une cible : la thrombine. C'est un modèle déjà étudié pour lequel deux aptamères ont fait l'objet de publications. Nous avons utilisé l'aptamère T29 dans le cadre de notre étude car c'est celui qui présente la meilleure affinité. L'électrophorèse capillaire est un puissant outil analytique qui facilite l'efficacité de sélection des aptamères et précise la détermination des paramètres d'interactions. Nous avons ainsi pu déterminer la constante d'affinité KD par CE-UV-LEDIF sur le modèle de base : la thrombine. Par ailleurs, nous montrons également comment l'utilisation du tampon Tris peut dégrader un ADN simple brin en électrophorèse capillaire et nous proposons comme alternative l'utilisation d'un tampon sodium phosphate dibasique qui évite ce phénomène de dégradation. Enfin, nous expliquons la difficulté d'amplification par qPCR et PCR d'un aptamère comme le T29 ayant une structure en G-quadruplex. Nous avons montré que le séquençage haut-débit Illumina nous a permis de trouver une corrélation entre le nombre de molécules séquencées et le nombre de séquences obtenues. L'analyse des séquences obtenues montre une quantité importante (20%) de séquences de T29 qui ne correspondent pas à la séquence de cet aptamère. Cela prouve que les étapes de PCR et de séquençage haut débit pour la détection de G-quadruplex peuvent induire un biais dans l'identification de ces molécules
Aptamers are oligomers of small single-stranded DNA or RNA which can have strong and specific interactions with some targets when they fold into three-dimensional structures. The objective of this thesis was to complete existing studies on the use of capillary electrophoresis in order to develop a method for the selection of aptamers by CE coupled to laser induced fluorescence and Illumina high-throughput sequencing. In a first step, we developed a method of detection and separation by capillary electrophoresis coupled with the double detection UV-LEDIF of a DNA library interacting with a target: thrombin. It is a model already studied and for which two aptamers have been published. We used aptamer T29 as part of our study because it has the best affinity. Capillary Electrophoresis is a powerful analytical tool that facilitates the selection efficiency of aptamers and specifies the determination of the interaction parameters. We thus were able to determine the affinity constant KD by CE-UV-LEDIF on the basic model: thrombin. Moreover, we also show how the use of Tris buffer can degrade single-stranded DNA during capillary electrophoresis and we propose as an alternative the use of a dibasic sodium phosphate buffer which avoids the phenomenon of degradation. Finally, we explain the difficulty of amplification by qPCR and PCR of an aptamer such as T29 with a G-quadruplex structure. We showed that the Illumina high-throughput sequencing allowed us to find a correlation between the number of sequenced molecules and the number of sequences obtained. Analysis of the sequences obtained shows a significant amount (20%) of T29 sequences which do not correspond to the sequence of this aptamer. This shows that the PCR and high-throughput sequencing steps for the detection of G-quadruplex can induce bias in the identification of these molecules

De nos jours, il est urgent de créer des biocapteurs peu coûteux, efficaces et simples à utiliser, capables de quantifier et de détecter rapidement des petites molécules (masse molaire <1000 g / mol) telles que les pesticides, les toxines, les antibiotiques ou les médicaments, pour protéger, à la fois la santé humaine et l’environnement. Les aptamères sont des oligonucléotides en série ARN ou ADN présentant une affinité et une spécificité élevées pour leur cible. Ils peuvent être utilisés comme éléments de reconnaissance moléculaire dans des biocapteurs, appelés aptacapteurs. Ces derniers offrent une alternative prometteuse aux méthodes conventionnelles pour la détection des petites molécules. L'objectif de cette thèse est de concevoir de nouveaux aptacapteurs spécifiques basés sur le couplage de deux stratégies : « light-up » et « kissing-complexe ». Dans un premier temps, une détection à base d'aptamères fluorescents spécifiques de deux précurseurs de microARN, let-7b et miR206, a été développée. Suivant ce concept, un aptacapteur fluorescent à double reconnaissance capable de détecter la théophylline a été par la suite créé. Enfin, deux approches utilisant la méthode d'anisotropie de fluorescence et le déplacement d’un brin complémentaire de l’aptamère ou d’un intercalant de l’ADN ont été appréhendées pour la détection de deux pesticides, l'isocarbophos et le phorate
Nowadays, there is an urgency to detect small molecules (molecular weight < 1000 g/mol) such as pesticides, toxins, antibiotics or drug residues to protect both human and environment health. It is very important to create a cost effective and simple to use sensor system able to rapidly quantify small molecules, with high efficiency of measurements. In this context, aptamers that are RNA or DNA oligonucleotides displaying high affinity and specificity for their cognate target can be used as molecular recognition element in biosensors. These biosensors called aptasensors can provide a valid and interesting alternative to conventional methods.The aim of this thesis consists in engineering new specific aptasensors based on the coupling of two strategies: light-up and kissing-complex. Firstly, we designed a fluorescent aptamer-based sensing specific of two microRNA precursors: let-7b and miR206. In a second time, based on this construct, we create a label free fluorescent double-switch aptasensor to detect theophylline. Finally, in the third work, two aptamer-based fluorescence anisotropy approaches, using the displacement concept, are investigated to detect pesticides including isocarbophos and phorate

Philosophiae Doctor - PhD
In this work, DNA aptamer biosensor systems were developed for the detection of l7p-estradiol - an estrogeneous endocrine disrupting chemical (EDC). Endocrine disrupting chemicals are group of compounds that impact negatively on the endocrine system of humans and wildlife. High concentrations of l7p-estradiol in water or food chain disrupts the physiology of the endocrine system of various animal species, leading to feminisation in fish and stimulates the proliferation of cancer cells in humans. Aptasensor systems for the determination of l7pestradiol were prepared with three immobilization platforms: (i) poly(3,4- ethylenedioxythiophene) {PEDOT} doped with gold nanoparticles (AuNPs) to form PEDOTIAuNPs polymeric nanocomposite, (ii) generation 1 poly(propylene thiophenoimine)-copoly( 3 ,4-ethy lenedioxythiophene) dendritic star copolymer (G 1PPT -co-PEDOT), and (iii) generation 2 poly (propylene thiophenoimine)-co-poly(3,4-ethylenedioxythiophene) dendritic star copolymer (G2PPT-co-PEDOT). The morphological properties of the sensor platforms were interrogated by scanning emission microscopy (SEM) and atomic force microscopy (AFM), while their spectroscopic characteristics were studied by Fourier transform infra red spectroscopy (FTIR) and fluorescence spectroscopy. The electrochemical behaviour of the platforms and the aptasensors were studied by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and square wave voltammetry (SWV). The DNA aptamer developed for detecting 17~-estradiol and which was used in the fabrication of all aptamer biosensors in this study is a 76-mer biotinylated aptamer (5'-BiotinGCTTCCAGCTTATTGAATTACACGCAGAGG TAGCGGCTCTGCGCATTCAATGCTGCGCGCTGAAGCGCGGAAGC-3'). AulPEDOTIAuNPslAptamer (platform 1) was obtained by covalently attaching streptavidin to the polymeric nanocomposite platform using carbodiimide chemistry and the aptamer immobilized via streptavidin-biotin interaction. The electrochemical signal generated from the aptamer-target molecule interaction was monitored electrochemically using cyclic voltammetry and square wave voltammetry in the presence of [Fe(CN)6J 3-/4- as a redox probe. The signal current observed was inversely proportional to the concentration of 17Bestradiol. The aptasensor demonstrated specificity toward 17~-estradiol. The detectable concentration range of the 17B estradiol was 0.01 nM-O .09 nM with a detection limit of 3.2 pM. The 76-mer biotinylated aptamer for 17~-estradiol was incorporated into a generation 1 poly(propylenethiophenoimine )-co-poly(3 ,4-ethylenedioxythiophene) dendritic star copolymer modified Au electrode via biotin-avidin interaction (platform 2). The Bode plot shows that the charge transfer dynamics of the nanoelectrode can be frequency modulated while the AulG 1PPTco- PEDOT nanoelectrode exhibited greater semi-conductor behavior (higher phase angle value) than AulG 1PPT due to the incorporation of charged functionalized dendrimer at low frequencies (100 mHz). The biosensor response to 17~-estradiol was based on the decrease in the SWV current as the EDC binds to the ssDNA aptamer on the biosensor. The dynamic linear range of the sensor was 0.01-0.07 nM with a detection limit of7.27 pM. Synthesis of electro synthetic generation G2PPT-co-PEDOT (platform 3) was performed by copolymerization of PEDOT with G2PPT dendrimer modified electrode immersed in a solution of 0.1 M LiCI04 containing 0.1 M EDOT monomer and 0.1 M sodium dodecyl sulphate (SDS) for ten (10) cycles. The electrochemical behaviour of the dendritic star copolymer was investigated with CV and EIS in LiCI04 and phosphate buffer solutions. The results show that the electrochemical deposition of G2PPT-co-PEDOT on gold electrode decreased the electrochemical charge transfer resistance when compared to AuiPEDOTILiCI04 and AuiLiCI04 interfaces. Bode impedimetric analysis indicates that G2PPT-co-PEDOT is a semiconductor. The fabrication of two novel aptasensors (based on platforms 2 and 3) simultaneously on a screen printed micro array electrode of 96-well multichannel electrochemical robotic sensor testing system for the detection of endocrine disrupting l7~-estradiol, was also carried out. The aptasensors responses to l7~-estradiol, based on the decrease in the SWV current, were evaluated.

Células tronco mesenquimais de tecido adiposo, são uma promissora ferramenta para aplicações clínicas em terapias celular e regenerativa, em vista da facilidade de sua extração e da maior quantidade de células por unidade de massa de tecido quando comparado a outras fontes clássicas de células mesenquimais como medula óssea. O protocolo clássico de extração e purificação dessas células, depende de sua adesão em plástico e xeno-materiais demandando muito tempo para ser utilizado por médicos para auxiliar pacientes em procedimentos de emergência. Estas células são capazes se diferenciar em diversos tipos celulares, o que as torna boas candidatas para terapia celular, embora sua capacidade de transdiferenciação para fenótipos neuronais seja ainda discutida. Neste trabalho demonstramos um novo processo para isolar essas células na base de epitopos específicos expressos (assinatura molecular de superfície) utilizando aptâmeros como ligantes de alta afinidade para estes sitios. Aptâmeros, moléculas de DNA simples fita identificadas a partir de uma biblioteca combinatória de sequencias de DNA simples-fita foram identificados por ciclos reiterativos de seleção in vitro (SELEX) utilizando células tronco do lipoaspirado como alvo. Dois aptâmeros isolados, denominados APT9 e APT11, foram capazes de identificar subpopulações (15,8 e 23,7% respectivamente) dentre as células tronco mesenquimais (classicamente CD29+/CD90+/CD45-) e separá-las usando nano-partículas magnéticas acopladas aos aptâmeros. Além disso, seguindo uma indução para diferenciação neuronal, as células tronco mesenquimais passam a apresentar morfologia neuronal e apresentam expressão e atividade de diversos receptores de neurotransmissores, avaliados por PCR real-time e imageamento de variações da concentração de cálcio intracelular ápos stimulação com vários agonistas de receptores metatrópicos e ionotrópicos. Ao longo da diferenciação, os níveis transcricionais de mRNA de receptores de cininas (B1 e B2), nicotínicos (alfa 7), muscarínicos (M1, M3 e M4), glutamatérgicos (AMPA2 e mGluR2), purinérgicos (P2Y1 e P2Y4) e GABAergicos (GABA-A, subunidade 3) e da óxido nítrico sintase neural aumentaram quando comparados aos níveis das células não diferenciadas, enquanto que os níveis de expressão de outros receptores incluindo purinérgicos P2X1, P3X4, P2X7 e P2Y6 e muscarínico M5 diminuíram. Os níveis de atividade das classes dos receptores estudados, por imageamento de variações da concentração de cálcio intrac, aumentaram para a maioria dos agonistas analisados durante a diferenciação neuronal com exceção para respostas induzidas por glutamato e NMDA. Células diferenciadas expressavam altos níveis de antígenos específicos de neurônios como β3-tubulina, NF-H, NeuN e MAP-2 indicando uma diferenciação em fenótipo neuronal bem sucedida. Desta maneira, esta tese, ao identificar aptâmeros, prove uma inovadora solução para médicos usarem as células tronco mesenquimais dentro de uma sala de cirurgia, através de um método que é capaz de purificar essas células em um tempo clínico viável, com pureza e sem contato com contaminantes. Além disso, nós mostramos aqui que com um protocolo como o proposto para diferenciação neuronal, nós poderíamos induzir essas células para se diferenciar em neurônios, através da ativação de fatores de transcrição específicos, levando às células tronco mesenquimais a serem possivelmente utilizadas em terapias celulares de reparo neuronal.
Adipose mesenchymal stem cells are promising tools for clinical applications in cellular and regeneration therapies, in view of easiness of extraction and higher amount of isolated stem cells per mass of tissue when compared to other classical mesenchymal stem cell sources including bone marrow. The classical protocol to extract and purify these cells, depending on plastic adherence and xeno-materials, is too time consuming to be used by physicians to help patients at emergency procedures. These cells are able to differentiate into various cell types, making them good candidates for cell therapy, however their capability for transdifferentiation into neural phenotypes is yet discussed. Here we show a novel process to isolate these cells using their surface molecular signature and aptamers, ssDNA molecules identified through the SELEX technique, denominated APT9 and APT11 that are able to identify subpopulations (15,8 and 23,7% respectively) within the mesenchymal stem cells (classically CD29+/CD90+/CD45-) and separate them using magnetic nano-particles attached to the aptamers. Moreover, following induction to neural differentiation, mesenchymal cells presents neuronal morphology and present expression and activity of several neurotransmitter receptors, as evaluated by real-time PCR and calcium imaging. During this process, mRNA transcription levels of bradykinin (B1 and B2), cholinergic (alpha 7), muscarinic (M1, M3 and M4), glutamatergic (AMPA2 and mGlu2), purinergic (P2Y1 and P2Y4) and GABAergic (GABA-A, subunit 3) receptors and neuronal nitric oxide synthase were augmented when compared to levels of undifferentiated cells, while the expression levels of other receptors including purinergic P2X1, P2X4, P2X7 and P2Y6 and muscarinic M5 receptors were down-regulated. Activity levels of the studied receptor classes, as studied by calcium imaging, increased for most of the agonists analyzed during the neuronal differentiation with the exception for glutamate- and NMDA-induced receptor responses. Differentiated cells expressed high levels of neuron-specific antigens such as β3-tubulin, NF-H, NeuN and MAP-2, indicating a successful differentiation into neuronal phenotypes. This thesis, by identifying aptamers, provides a novel solution for physicians to use mesenchymal stem cells inside a surgery room, by using a method that are able to purify the cells in a clinical viable time, with purity and no contact with contaminats. Furthermore, we show here that with a protocol as provided for neuronal differentiation, we could induce these cells to differentiate into neurons, by activating specific transcription factors,making mesenchymal stem cells to possibly be used in neuronal repair cell therapies.

Fur (Ferric Uptake Regulator) est un régulateur transcriptionnel spécifique des bactéries qui intervient dans le contrôle de l'homéostasie du fer, ce qui en fait une cible antibactérienne intéressante. Avant mon arrivée au laboratoire, quatre inhibiteurs interagissant spécifiquement avec Fur avaient été isolés. La partie active de ces inhibiteurs consiste en des peptides de 13 acides aminés. Au cours de cette thèse, j'ai utilisé une double-approche : théorique et expérimentale pour étudier l'interaction de ces peptides avec Fur afin de comprendre le mécanisme d'inhibition. J'ai synthétisé plusieurs séquences peptidiques, montré par des tests biochimiques que certaines inhibaient Fur et déterminé les interactions importantes à l'activité inhibitrice. J'ai obtenu des modèles théoriques des complexes Fur/peptides par amarrage moléculaire, cohérents avec les résultats expérimentaux, qui ont mis en évidence une zone d'inhibition de Fur. Des criblages in silico dans cette zone ont permis de sélectionner de petites molécules, inhibitrices potentielles de Fur et donc intéressantes pour des applications thérapeutiques.

Les aptamères sont des acides nucléiques capables de se lier sélectivement à un ligand ou à une famille de molécules. Les aptamères sont la partie sensible des riboswitches, qui sont des segments régulateurs de l'ARN messager impliqués dans l'expression génétique. Les aptamères ont aussi des applications prometteuses comme sondes artificielles et capteurs Pour ces technologies, il est crucial de comprendre comment la liaison se produit, de la quantifier, et de comprendre comment les changements conformationnels sont induits par les ligands. Les objectifs de cette thèse sont d'explorer l'applicabilité de la spectrometrie de mobilité ionique (IMS) couplée à la spectrométrie de masse (SM) native aux aptamères d'ADN et d'ARN, d'abord dans la quantification de liaison, ensuite dans la détection du changement conformationnel lors de la liaison du ligand.Dans la première partie, nous avons évalué la détermination des valeurs de constantes d’équilibre de dissociation (KD) par MS, en tenant compte des facteurs de réponse relatifs (Rx) des aptamères libres et liés. Les titrages en SM sont comparés, pour validation, avec la calorimétrie par titrage isotherme (ITC). Deux aptamères d'ARN sont pris comme modèles : l'aptamère du vert de malachite, largement étudié par ITC, et l'aptamère de la riboflavine mononucléotide , un cas réaliste d'ARN Mg2+-dépendant pour la liaison du ligand. Nous avons observé que l'acétate d'ammonium et l'acétate de triméthyl ammonium conviennent à l'étude des aptamères et leurs complexes, et que les valeurs de KD obtenues par ITC et SM native sont comparables. Les aptamères ARN de la néomycine et de la tobramycine ont été choisis pour tester la limite de détection en SM native. Nous concluons que la SM native est adaptée pour déterminer des valeurs de KD comprises entre 50 nM et 30 µM. La correction apportée par Rx est relativement modeste dans tous les cas, en suggerant que la liaison du ligand n'est pas associée à une différence conformationnelle significative lors de l'ionisation. Pour ces aptamères, nous concluons que l'hypothèse de Rx égaux est acceptable.Dans la deuxième partie, nous avons évalué si le mécanisme de "liaison adaptative" des aptamères peut être révélé par IMS. À cette fin, en plus des systèmes énumérés ci-dessus, nous avons étudié l'aptamère ARN de la tétracycline et une série d'aptamères ADN capables de lier la cocaïne, pour lesquels le changement conformationnel par liaison du ligand est largement documenté dans la littérature. Pour tous les aptamères à l'exception de l'aptamère de la tétracycline, nous n'avons pas observé de différences significatives dans la conformation en phase gazeuse des ions liés aux ligands ou Mg2+. Cependant, nous avons observé un changement significatif dans la mobilité des ions de l'aptamère de la tétracycline. Le Mg2+ (100 µM) s’avère essentiel pour la liaison du ligand. Pour la série des aptamères de la cocaïne, même si nous ayons observé dans des conditions douces de pré IMS des ions compacts aussi bien pour les aptamères libres que pour les aptamères liés, une extension conformationnelle est visible à haute activation pre-IMS, bien révélée par l'état de charge 7-, qui suggère des réarrangements de phase gazeuse. Pour mieux étudier ces réarrangements, nous avons modifié les séquences avec des extensions dA, afin de comparer des systèmes ayant un nombre similaire de degrés de liberté sans modifier la structure cœur. Nous proposons également de nouvelles façons de présenter ces données, mieux adaptées quand la dissociation du ligand, la perte d’aduits et le dépliement d’ion arrivent dans les mêmes gammes d’énergie. L'augmentation graduelle de l'activation collisionnelle avant l'IMS, a révélé que l’energie de dépliement est corrélée au contenu en paires de bases, ce qui suggère que les paires de bases sont conservées dans les structures en phase gazeuse. Nous avons également observé que le ligand se perd à des énergies inférieures à celles du dépliage
Aptamers are single-stranded nucleic acids capable to bind selectively to a ligand or to a family of molecules. Aptamers are the sensing part of riboswitches, which are regulatory segments of messenger RNA involved in gene expression. Aptamers are also promising artificial probes, sensors and stimuli-responsive elements. In the development of aptamer-based technology, it is crucial to understand how binding is occurring, to quantify affinities, and ligand-induced conformational changes. The objective of this thesis is to explore the applicability of native IM-MS to DNA and RNA aptamers to quantify binding and to detect conformational change upon binding.In the first part, we evaluated the quantitative determination of equilibrium dissociation constants (KD) by mass spectrometry (MS), and the necessity of including a correction for relative response factors of free and bound aptamers. We compared isothermal titration calorimetry and MS titrations to validate the quantifications. Two RNA aptamers were taken as models: the malachite green aptamer, extensively studied by ITC, and the riboflavin mononucleotide aptamer, a case of Mg2+-dependent ligand binding. We observed that typical volatile electrolytes ammonium acetate and trimethyl ammonium acetate are suitable to study RNA aptamer binding, and that comparable KD values are obtained from ITC and native MS. The neomycin and tobramycin RNA aptamers were chosen to test the limit of detection of native MS. We found that native MS is appropriate to determine KD values in the range from 50 nM to 30 µM. The relative response factor correction was relatively modest in all cases, suggesting that the ligand binding is not associated to a significant conformational difference upon ionization. For these aptamers, we conclude that assuming equal response factors is acceptable.In the second part, we evaluated whether the aptamers’ “adaptive binding” mechanism can be revealed by ion mobility spectrometry (IMS). To this aim, in addition to the systems listed above we studied the tetracycline RNA aptamer and a series of cocaine-binding DNA aptamers, for which the conformational change upon binding is reported in literature. For all aptamers except the tetracycline aptamer, we did not observe a significant difference in the shape of the gas-phase structure upon ligand or Mg2+ binding. However, a significant change was observed in tetracycline RNA aptamer’s ion mobilities, at biologically relevant concentration of Mg2+ (100 µM), and we found that Mg2+ is essential for ligand binding, in agreement with previous solution studies. For the cocaine-binding DNA aptamer series, although we observed similar compactness for the free and bound aptamers in soft pre-IMS conditions, a conformational extension occurs at high pre-IMS activation, best revealed by charge state 7-, suggesting gas-phase rearrangements. To better investigate whether the energetics of these rearrangements depend on pre-folding or on ligand binding, we modified the sequences with dA overhangs, to compare systems with similar numbers of degrees of freedom without altering the core structure. We also propose new ways of presenting the data, adapted to the cases where ligand dissociation, declustering and unfolding occur at similar voltages. The gradual increase of the pre-IMS collisional activation revealed that the unfolding energetics is correlated with the base pairs content, suggesting that base pairs are conserved in the gas-phase structures. We also found that ligand is lost at lower energies than unfolding.In summary, gas-phase compaction occur for both the free aptamers and bound aptamers, and memories of the solution-phase structures can only be revealed in some particular cases. However, the compaction towards similar shapes might constitute an advantage for the quantification, because molecular systems of similar shapes have similar electrospray responses. Consequently, native MS provides reliable estimations of KD values

Cyanotoxins are posing an increasing threat to the health of humans and wildlife. Cylindrospermopsin is a cyanotoxin that occurs in warm climates and is harmful when ingested. The toxic effects of CYN can affect multiple organ systems. The effects, coupled with the evidence of a mass contamination of a water supply in Australia, prove that CYN needs to be investigated further. Aptamers have become a desirable method for detection of CYN as a result of an aptamer’s high specificity and the ability to scale up experiments. Aptamers have been designed to bind with a variety of targets, including cyanotoxins. An aptamer for CYN was identified by Elshafey et al. This study aims to confirm the binding of the aptamer to CYN and the selectivity of the aptamer using fluorescent biosensing and circular dichroism. Aptamer affinity capture was used to investigate the possibility of a real world application of the aptamer.

[ES] La presente tesis doctoral titulada "New nanostructured suports with signal amplification features for the detection of molecules and biomolecules of interest" se centra en el diseño y preparación de nuevos materiales híbridos orgánicos-inorgánicos constituidos por puertas moleculares soportadas sobre alúmina mesoporosa con el objetivo de desarrollar nuevos sistemas sensores con aplicaciones potenciales en el campo de la diagnosis y del control alimentario. En el primer capítulo de la tesis se introducen los conceptos en los que están basados los estudios realizados y los materiales preparados. A continuación, en el segundo capítulo se describen los objetivos generales de la tesis que serán abordados en los siguientes apartados. En el tercer capítulo se presenta el diseño y optimización de un nanodispositivo para la detección de la bacteria Mycoplasma fermentans. En primer lugar, los poros de una placa de alúmina mesoporosa se cargan con un indicador fluorescente (rodamina B). Seguidamente, la superficie es funcionalizada con una secuencia de ADN complementaria a una región altamente conservada de la subunidad ribosomal 16S de la bacteria Mycoplasma fermentans. El impedimento estérico generado por las secuencias de ADN ancladas al exterior de los poros impide la salida del indicador encapsulado. Únicamente en presencia de DNA de la bacteria Mycoplasma fermentans, se produce la apertura de los poros permitiéndose la difusión de la carga (rodamina B) que es posteriormente medida mediante espectroscopía de fluorescencia. En el capítulo cuatro se diseña de un nanodispositivo capaz de detectar de forma rápida, sensible y selectiva la bacteria Staphylococcus aureus. Para la preparación del material sensor, un soporte de alúmina mesoporosa es, en primer lugar, cargado con el indicador fluorescente rodamina B. A continuación, los poros del soporte son tapados mediante el anclaje de un aptámero que reconoce de forma específica la bacteria. Solamente en presencia de Staphylococcus aureus se produce la liberación del indicador encapsulado, que es posteriormente medido mediante espectroscopía de fluorescencia. Además, la respuesta obtenida es específica para Staphylococcus aureus. Este sistema ha sido ensayado en muestras reales. En el sexto capítulo, diseña un nanodispositivo híbrido orgánico-inorgánico consistente en un material de alúmina mesoporosa cubierto con una secuencia de ADN específica para la detección de ADN del hongo Pneumocystis jirovecii. En este caso, el soporte de alúmina cargado con rodamina B se recubre con una secuencia de ADN específica para el reconocimiento de este hongo. En presencia del organismo, la horquilla hibrida con el ADN del hongo, lo que resulta en una conformación triplex con elevada afinidad y estabilidad que induce, al mismo tiempo, el desplazamiento de este complejo de la superficie. Como consecuencia de este reconocimiento la carga se libera y es cuantificada mediante espectroscopía de fluorescencia. El sistema ha sido satisfactoriamente validado. En el séptimo capítulo, se diseña un sistema sensor con la capacidad de detectar gluten de forma rápida y sencilla en extractos de alimentos procesados y no procesados. Para ello, un soporte de alúmina mesoporosa se carga con rodamina B y los poros se recubren con un aptámero específicamente diseñado para la detección de la proteína gliadina, que constituye el 50 % del total del clúster de elementos que forman el gluten. La elevada afinidad y especificidad entre el aptámero y la proteína en cuestión hacen que en presencia de ésta se produzca un desplazamiento de la puerta molecular que permite la difusión del colorante encapsulado que es finalmente monitorizado mediante espectroscopía de fluorescencia. Finalmente, en el capítulo octavo se discuten de forma conjunta los resultados obtenidos en los capítulos anteriores y la potencial aplicación de los sistemas desarrollados en el actual sistem
[CA] La present tesi doctoral, titulada "New nanostructured supports with signal amplification features for the detection of molecules and biomolecules of interest", es centra en el disseny i preparació de nous materials híbrids orgànics-inorgànics constituïts per portes moleculars suportades sobre alúmina mesoporosa amb l'objectiu de desenvolupar nous sistemes sensors amb potencials aplicacions en el camp de la diagnosi i del control alimentari. En el primer capítol de la tesi s'introdueixen els conceptes en què estan basats els estudis realitzats i els materials preparats. A continuació, en el segon capítol es descriuen els objectius generals de la tesi que seran abordats en els següents apartats. En el tercer capítol es presenta el disseny i optimització d'un nanodispositiu per a la detecció de la bactèria Mycoplasma fermentans. Primerament, els porus d'una placa d'alúmina mesoporosa són carregats amb un indicador fluorescent (rodamina B). Seguidament, la superfície és funcionalitzada amb una seqüència d'ADN complementaria a una regió altament conservada de la subunitat ribosomal 16S de la bactèria Mycoplasma fermentans. L'impediment estèric generat per les seqüències d'ADN ancorades a l'exterior dels porus impedeix l'alliberament de l'indicador encapsulat. Únicament en presencia d'ADN de la bactèria Mycoplasma fermentans, es produeix l'obertura dels porus permetent la difusió de la càrrega (rodamina B) que és posteriorment mesurada mitjançant fluorescència. En el capítol quatre es dissenya un nanodispositiu capaç de detectar de forma ràpida, sensible i selectiva la bactèria Staphylococcus aureus. Per a la preparació del material sensor, el suport d'alúmina mesoporosa és, primerament, carregat amb l'indicador fluorescent rodamina B. A continuació, els porus del suport són tapats mitjançant l'ancoratge d'un aptàmer que reconeix de forma específica a la bactèria. Solament en presència de Staphylococcus aureus es produeix l'alliberament de l'indicador encapsulat, que és posteriorment mesurat mitjançant espectroscòpia de fluorescència. A més a més, la resposta obtinguda és específica per Staphylococcus aureus. Aquest sistema ha sigut validat amb mostres reals de pacients. En el sisè capítol, es dissenya un nanodispositiu híbrid orgànic-inorgànic consistent en un material d'alúmina mesoporosa cobert amb una seqüència d'ADN específica per a la detecció de l'ADN del fong Pneumocystis jirovecii. En aquest cas, el suport d'alúmina carregat amb l'indicador fluorescent rodamina B és recobert amb una seqüència d'ADN específica per al reconeixement d'aquest fong. En presència de l'organisme, la forquilla hibrida amb l'ADN del fong, resultant en una conformació triplex amb elevada afinitat i estabilitat, que indueix, al mateix temps, el desplaçament d'aquest complex de la superfície. Com a conseqüència d'aquest reconeixement la càrrega és alliberada i quantificada mitjançant espectroscòpia de fluorescència. El sistema ha sigut validat com a mètode diagnòstic mitjançant l'anàlisi de mostres reals de pacients. En el seté capítol, es dissenya un sistema sensor amb la capacitat de detectar gluten de forma ràpida i senzilla en extractes d'aliments processats i no processats. Per a això, un suport d'alúmina mesoporosa es carrega amb indicador fluorescent rodamina B i posteriorment és recobert amb un aptàmer específicament dissenyat per a la detecció de la proteïna gliadina, que constitueix el 50 % del total del clúster d'elements que formen el gluten. L'elevada afinitat i especificitat entre l'aptàmer i la proteïna en qüestió fa que en presència d'aquesta es produesca un desplaçament de la porta molecular que permet la difusió de la càrrega encapsulada i que serà finalment monitoritzada mitjançant espectroscòpia de fluorescència. Finalment, en el capítol vuité es discuteixen de manera conjunta els result
[EN] The PhD thesis hereby presented and entitled "New nanostructured supports with signal amplification features for the detection of molecules and biomolecules of interest", focuses in the design and preparation of new hybrid organic-inorganic materials constituted by molecular gates supported over mesoporous alumina with the aim of developing new sensor probes of potential applications in the fields of diagnosis and food control. In the first chapter, the concepts in which studies and prepared materials are based, are introduced. Next, the second chapter describes the general objectives of this thesis, which will be approached in the following sections. In the third chapter, it is presented in detail the design and optimization process of a nanodevice applied for the detection of Mycoplasma fermentans bacterium. First of all, mesoporous alumina porous films are charged with a fluorescent indicator (rhodamine B). Then, the surface is functionalized with a DNA sequence complementary to a highly conserved region of the 16S ribosomal subunit of the bacterium Mycoplasma fermentans. Steric hindrance generated by DNA sequences on the surface inhibits the release of the encapsulated indicator. Only in the presence of bacterium Mycoplasma fermentans DNA, molecular gates open, allowing payload diffusion to the solution, which is measured by fluorescence spectroscopy. In chapter four, it is carried out the design and optimization of a nanodevice able to detect Staphylococcus aureus bacterium in a fast, sensitive and selective way. For the sensor preparation, alumina mesoporous support is, first, loaded with the rhodamine B fluorescent dye. Then, the mesoporous are blocked through the attachment of an aptamer that recognises specifically this bacterium. Exclusively in the presence of Staphylococcus aureus it is accomplished the release of the encapsulated dye, which is later monitored by fluorescence spectroscopy. The response obtained is specific for Staphylococcus aureus. This system has been validated in real samples. In the sixth chapter, it is detailed the design and optimization process of a hybrid organic-inorganic nanodevice based on a capped mesoporous alumina material for the detection of Pneumocystis jirovecii fungus DNA. In this case, the mesoporous alumina support is loaded with a fluorescent dye and decorated with a specific oligonucleotide sequence designed for the recognition of Pneumocystis fungus. In the presence of the target organism, the fork-like oligonucleotide hybridises with the DNA of the fungus, which results in the adoption of a triplex conformation with high affinity and stability that induces, at the same time, the displacement of this complex from the surface. Consequently, the payload diffused to the solution is quantified through fluorescence spectroscopy. The system has been successfully validated. In the seventh chapter, it was developed a sensor system for gluten detection, in a quick and easy way, in processed and non-processed food extracts. For this, a mesoporous alumina support is loaded with the fluorescent dye rhodamine B, and later was functionalized with an aptamer specifically designed for the detection of gliadin, a protein that constitutes 50 % of average cluster elements that forms gluten. The protein-aptamer high affinity and specificity induce the displacement of the capping aptamer and cargo delivery, which is monitored through fluorescence spectroscopy. Finally, in the eighth chapter, the results obtained in the previous chapters and the potential application of the systems developed as health and food control system are discussed.
We thank the Spanish Government projects MAT2015-64139-C4-1-R, AGL2015-70235-C2-2-R, and TEC2015-71324-R (MINECO/FEDER, UE), the Generalitat Valenciana (project PROMETEOII/2014/047), the Catalan authority (project AGAUR 2014SGR1344), and ICREA under the 2014 ICREA Academia Award for support. This study was supported by the Spanish Government projects RTI2018-100910-B-C41 and SAF2017-82251-R (MCUI/AEI/FEDER, UE), the Generalitat Valenciana (project PROMETEO/2018/024), the Universitat Politècnica de València−Instituto de Investigación Sanitaria La Fe (B02-MIRSA project), CIBER-BBN (NANOPATH and valorization project CANDI-EYE) and co-financed by the EU through the Valencian Community ERDF PO 2014-2020. This research was funded by the Spanish Government, projects RTI2018-100910-B-C41 (MCUI/AEI/FEDER, UE) and CTQ2017-84415-R
Pla Blasco, L. (2021). New nanostructured supports with signal amplification features for the detection of molecules and biomolecules of interest [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/166500
TESIS

Aptamers are short single-stranded DNA or RNA oligonucleotides artificially selected against specific targets. They exhibit high target binding affinity and exquisite specificity, making them very useful in developing biosensors for a wide range of targets, such as proteins, peptides, amino acids, drugs, metal ions and even whole cells. While fluorescent semiconductor nanocrystals, also known as quantum dots (QDs), have unique size-dependent, bright and extremely photo-stable fluorescence that make them as excellent fluorescent labels for biological imaging, sensing, cell tracking/trafficking and diagnostics. Their unique optical properties (broad absorption and narrow symmetric emission) are well-suited for FRET (fluorescence resonance energy transfer) based sensing applications. By combining the advance properties of both aptamers and QDs, this project aims to develop a sensitive, specific and robust aD-DNAlaptamer FRET based biosensing technology that can be used for rapid biosensing, diagnostics and environmental monitoring. A major hurdle here is the preparation of a compact, stable and water-soluble QD-bioconjugate that can effectively resist non-specific adsorption because FRET efficiency (E) decreases dramatically with the increasing donor-acceptor distance. Hence for high sensitivity, a compact QD-bioconjugate structure is essential. In this regard, a series of highly fluorescent, water-soluble CdSe/ZnS aDs were prepared first by ligand exchange with hydrophilic thiolated ligands, such as dihydrolipoic acid (DHLA), glutathione (GSH), dihydrolipoic acid-polyethyleneglycol (DHLAPEG600) derivatives. These QDs exhibited high fluorescence quantum yields (QYs, 6-30%), comparable to commercial water-soluble aDs (ca. 30%), but having significantly smaller hydrodynamic diameters «10 v.s. > 25 nm). Building upon these, three different QD-DNA aptamer sensing systems have been developed successfully: (1) A simple self-assembled aD-DNA system: I have found that thiolated DNA can self-assemble onto DHLA capped QDs to form compact, functional QD-DNA conjugates with small donor-acceptor distances, producing efficient FRET (E > 70%) at a relatively low (target: QD) copy number of 6:1. The resulting self-assembled aD-DNA (aptamer) conjugate has been used to detect low nM levels of labelled DNA target via QD sensitised dye FRET signal. Moreover, it has been successfully used for detection of nM level of a protein target via the encoded DNA aptamer sequence, although its specificity and stability still need further improvement. (2) A more stable and sensitive aD-dual-donor FRET sensing system based on an amine-modified DNA covalently coupled to a glutathione capped aD combined with the use of specific ethidium bromide (EB) intercalation in hybridized DNA duplex. As a result, both the aD and intercalated EBs can FRET to the dye acceptor appended to the complementary DNA, leading to significantly improved the overall FRET efficiency E, and hence sensitivity in both DNA and protein target detection down to pM level. (3) A Cu-free "clicked", robust, and_specific QD-DNA aptamer sensor. A compact, functional aD-DNA conjugate was prepared via the Cu-free "click chemistry" (CFCC) between a dihydrolipoic acid -polyethylene glycol-azide (DHLA-PEG600-N3) capped aD and a cyclooctyne modified DNA. The resulting QD-DNA conjugate is highly stable in biological buffer, effectively resisting nonspecific absorption, displaying a relatively small size (hydrodynamic radius - 5 nm) and retaining almost the native ay of the parent aD. Moreover, the CFCC based DNA conjugation method is also highly efficient, leading to high DNA loading (- 15-30 DNA strands per aD is readily achieved). This system is well-suited for robust biosensing: it can quantitate pM level of complementary DNA targets with SNP (singlenucleotide polymorphism) discrimination ability in complex media, e.g. 10% human serum. It can also detect pM level of a specific protein via the encoded DNA aptamer sequence. Compared with these approaches, the self-assembled system is the most convenient to prepare, but it has the least stability and cannot resist nonspecific absorption. The dual-donor FRET sensing system shows some enhancement on the stability and resisting nonspecific absorption, but it still cannot work in complex media, such as serum. The CFCC clicked QD-DNA aptamer sensor shows the highest stability, specificity and assay robustness, and can effectively work in clinical media. It can be readily extended to design sensors against other targets by simply clicking on specific aptamer sequences against such targets. Because the CFCC clicked QD-DNAlaptamer sensor shows high stability, specificity, robustness· and sensitivity, it may have a wide range of biosensing and diagnostic applications

Le mélanome est un cancer qui représente la grande majorité de la morbidité et de la mortalité causées par tout cancer de la peau en raison de son implication dans les métastases. Parmi plusieurs biomarqueurs rapportés, les récepteurs du domaine de la discoïdine (DDR) et la métalloprotéinase Matrix (MMP) sont également considérés comme des acteurs potentiels du mélanome. Des études récentes démontrent que les DDR et les MMP sont surexprimés dans les mélanomes de mauvais pronostic, ce qui nécessite le recours à des sondes théranostiques à biomarqueurs spécifiques. De nombreux progrès ont été réalisés pour réduire le risque associé au mélanome métastatique à l'aide d'anticorps monoclonaux traditionnels, mais les inconvénients tels que la complexité de la production, la variabilité d'un lot à l'autre, la courte durée de conservation, l'immunogénicité, la réactivité croisée et le coût élevé ne peuvent être négligés. Par conséquent, au cours des dernières années, les aptamères ont acquis un grand intérêt comme substitut d’anticorps pouvant être synthétisés chimiquement avec un faible coût de production et dépassant les limitations associées aux anticorps. Les aptamères sont de courts oligonucléotides à haute affinité et spécificité vis-à-vis de la molécule cible, développés par une méthode combinatoire appelée SELEX (Evolution systématique de ligands par enrichissement exponentiel). Au cours de ma thèse, nous avons réalisé une étude SELEX contre les biomarqueurs DDR1, DDR2 et HMMP14 sur la base de supports alternatifs pour le tri des candidats et le séquençage à haut débit. Un groupe d'aptamères a été sélectionné contre DDR1 et le meilleur candidat a ensuite été dopé pour effectuer davantage de tours de sélection contre DDR1. Finalement, un aptamère contre DDR1 a été sélectionné avec un Kd dans la gamme nanomolaire basse. Cet aptamère peut être utilisé comme outil théranostique dans les études sur le mélanome associé à DDR1. Comme les aptamères présentent un avantage en termes de conjugaison et de modifications aux positions désirées, nous visons en outre à conjuguer cet aptamère avec des nanoparticules pour des études in vivo
Melanoma is a cancer that accounts for the vast majority of morbidity and mortality caused by any skin cancer due to its involvement in metastasis. Among several reported biomarkers, Discoidin Domain Receptors (DDRs) and Matrix metalloproteinase (MMPs) are also considered as potential actors in melanoma. Recent studies demonstrate that DDRs and MMPs are overexpressed in melanoma with poor prognosis which demands the need of biomarkers specific theranostics probes. Though many advances has been made in order to reduce the risk associated with metastatic melanoma with the help of traditional monoclonal antibodies, but the drawbacks like production complexity, batch to batch variability, short shelf life, immunogenicity, cross reactivity and high cost cannot be neglected. Therefore in recent years, aptamers have gained high interests as a substitute of antibodies that can be synthesized chemically with low production cost and overcomes the limitations associated to antibodies. Aptamers are short oligonucleotides with high affinity and specificity against its target molecule, raised by a combinatorial method known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment). During my thesis, we performed a SELEX against DDR1, DDR2 and HMMP14 biomarkers based on alternative supports for candidates sorting and high throughput sequencing. A group of aptamers were selected against DDR1 and the best candidate was further doped to perform more rounds of selection against DDR1. Finally an aptamer against DDR1 was selected with a Kd in low nano-molar range. This aptamer can be used as a theranostics tool in DDR1 associated melanoma studies. As aptamers have an advantage of conjugation and modifications at desired positions so we further aim to conjugate this aptamer with nanoparticles for In-vivo studies

In the last years, DNA arrays have attracted increasing attention among medical diagnosis and analytical chemists. The broad range of application that has been found for DNA arrays makes them an important analytical tool. DNA arrays are relevant for the diagnosis of genetic diseases, detection of infectious agents, study of genetic predisposition, development of a personalised medicine, detection of differential genetic expression, forensic science, drug screening, food safety and environmental monitoring.
Despite the great promise of DNA arrays in health care and their success in medical and biological research, the technology is still far away from the daily use in the clinic and even more far away from their implementation in home-diagnosis such as glucose biosensors.
Their principal problems are the high cost and difficulty of use, because it is required costly laboratory instruments and biology knowledge for the labelling of the DNA prior to the sample injection into the array.
On the other hand, the requirements that a biosensor should include are to be easy-to use so that it do not need the previous label of the sample and the addition of reagents. It should give a sensitive response in short time, and it should also include cheap generic multi-analyte detection.
The work carried out in this thesis describes new concepts of electrochemical biosensoric platforms based on oligonucleotides for detection of label-free DNA and protein, which include these requirements.
Preliminary experiments of direct DNA electrochemical detection of labelled ssDNA were performed to establish a protocol of DNA immobilisation, hybridisation and detection colourimetrically and electrochemically. DNA real samples and multi-analite detection on an array developed by biocopatible photolithography were used.
To avoid the analyte labelling to develop an easy to use and low cost device, a label-free electrochemical displacement of DNA sensor was described. The method of detection by displacement requires the pre-hybridisation of the capture probe immobilised on the electrode surface with a sub-optimum mutated oligonucleotide labelled with a redox molecule. Due to the higher affinity of the target that is fully complementary to the capture probe, the sub-optimum label can be displaced when the complementary target is introduced in the system. The decrease of the signal would verify the presence of the target and should be proportional to its concentration.
Sub-optimum hybridisation displacement detection was demonstrated colourimetrically and electrochemically with a sub-optimum mutated oligonucleotide labelled with horseradish peroxidase (HRP), and a ferrocene sub-optimum mutated oligonucleotide was also detected electrochemically, which do not required the addition of reagents for its detection.
Furthermore different strategies to develop an electrochemical oligonucleotide (aptamer) based sensor for reagentless and label-free protein detection was carried out. The most sensitive aptasensor achieved 30 fM of detection limit in just 5 minutes.
En els últims anys, els xips d'ADN han atret una atenció creixen en els camps de la diagnosis mèdica i la química analítica, degut a la seva portabilitat, sensibilitat, especificitat, ràpida resposta i l'ampli ventall d'aplicacions. Els xips d'ADN són rellevants per la diagnosis de malalties genètiques, detecció d'agents infecciosos, estudis de predisposició genètica, desenvolupament de medicina personalitzada, detecció d'expressió genètica diferencial, medicina forense, exploració de medicaments, seguretat alimentaria, defensa militar i monitorització mediambiental.
Encara que els xips basats en oligonucleòtids per la detecció d'ADN i proteïnes siguin una gran promesa en medicina i recerca biològica, aquesta tecnologia es encara molt lluny del seu ús diari en el camp clínic i encara més lluny de poder ser comercialitzada per ús domèstic com ho han estat el biosensors de glucosa.
Els seus principals problemes són el seu alt cost i la seva dificultat d'ús. Ja que per la seva utilització és necessari, previ a la injecció de l'analit en el biosensor, costosos instruments de laboratori i tècnics especialitzats en bioquímica pel marcatge i amplificació de les mostres d'ADN.
En canvi els requeriments que un biosensor ha d'incloure són, ser fàcil d'utilitzar, per tant que l'analit no necessiti un marcatge previ i l'addició de reactius per la seva detecció. Aquest ha de donar una resposta ràpida i sensible a baix cost i ha de permetre la detecció en el mateix equip de diferent tipus d'analits.
El treball fet en aquesta tesis descriu el desenvolupament de nous concepte de plataformes biosensòriques electroquímiques basades en oligonucleòtids per la detecció d'ADN i proteïnes no marcades prèviament, els quals inclouen aquest requeriments.
Experiments preliminars per la detecció de l'hibridació d'ADN marcat es van portar a fi per tal d'establir un protocol per la immobilització, hibridació i detecció d'ADN colorimètricament i electroquímicament. És van utilitzar mostres reals d'ADN i sistemes de detecció de multi-analits en un xip desenvolupat per fotolitografia biocompatible.
Per tal de no necessitar un marcatge previ de la mostres d'ADN i així simplificar i reduir el cost del futur biosensor es va desenvolupar un sistema electroquímic de desplaçament. El mètode lliure de marcatge es basa en el desplaçament de molècules d'oligonucleòtid mutat i marcat, els quals encara que continguin certes mutacions són capaços d'hibridar amb la sonda d'oligonucleòtid immobilitzat, però quan aquestes es troben en presència de l'analit desplaça la molècula mutada i marcada, disminuint així la senyal de manera proporcional en la concentració del analit. El sistema de desplaçament ha estat demostrat colorimètricament i electroquímicament utilitzant un marcatge d'HRP sobre el mutat i utilitzant un marcatge de ferrocè en l'oligonucleòtid mutat per tal de no necessitar afegir cap reactiu per la detecció de l'analit,
També es van portar a fi diferents estratègies per desenvolupar un biosensor electroquímic basat en oligonucleòtids (aptamers) per la detecció de la proteïna trombina sense el previ marcatge d'aquest analit i sense necessitat d'afegir reactius per la detecció del analit. En el sistema mes sensible es va obtenir un límit de detecció de 30 fM en un temps de resposta de sols 5 minuts.
En los últimos años, los chips de ADN han atraído una atención creciente diferentes campos, debido a su portabilidad, sensibilidad, especificidad y rápida respuesta. Los chips de ADN son aplicados en diagnosis de enfermedades genéticas, detección de agentes infecciosos, estudios de predisposición genética, desarrollo de medicina personalizada, detección de expresión genética diferencial, medicina forense, exploración de medicamentos, columnas de separación, seguridad alimentaría, defensa militar y monitorización medioambiental.
Aunque los chips basados en oligonucleótidos para la detección de ADN y proteínas tienen un gran futuro en diagnosis e investigación biológica, esta tecnología está aun muy lejos de su uso diario en el campo clínico y aun mas lejos de poder ser comercializado para uso doméstico como lo han sido los biosensores de glucosa.
Sus principales problemas son su alto coste y su dificultad de uso. Para su utilización es necesario, previo a la inyección del analito en el biosensor, costosos instrumentos de laboratorio y técnicos especializados en bioquímica para el marcaje y amplificación de las muestras de ADN.
En cambio los requerimientos que un biosensor ha de incluir son, ser fácil de utilizar, por tanto el analito no ha de necesitar un marcaje previo ni la adición de reactivos para su detección. Este ha de dar una respuesta rápida y sensible a bajo coste y ha de permitir la detección en el mismo equipo de diferentes analitos.
El trabajo hecho en esta tesis describe el desarrollo de nuevos conceptos de plataformas biosensóricas electroquímicas basadas en oligonucleótidos para la detección de ADN y proteínas no marcadas previamente, los cuales incluyen estos requerimientos.
Experimentos preliminares para la detección directa de la hibridación de ADN marcado se llevó a cabo para establecer protocolos para la inmovilización, hibridación y detección de ADN colorimétricamente y electroquímicamente. Se utilizaron muestras reales y sistemas de detección de multi-analitos en un chip desarrollado por fotolitografía biocompatible.
Para no necesitar un marcaje previo de la muestra de ADN y así simplificar y reducir el coste del futuro biosensor se desarrolló un sistema electroquímico de desplazamiento. El método libre de marcaje se basa en el desplazamiento de moléculas de oligonucleótido mutado y marcado, el cual aunque contenga ciertas mutaciones es capaz de hibridar con la sonda de oligonucleótido inmovilizado, pero cuando estas se encuentran en presencia del analito desplaza la molécula mutada, disminuyendo así la señal de manera proporcional a la concentración del analito. El sistema de desplazamiento ha sido demostrado colorimétricamente y electroquímicamente utilizando marcaje de HRP sobre el mutado, así como un marcaje de ferroceno que no requiere la adición de reactivos para su detección.
También se llevaron a cabo diferentes estrategias para desarrollar un biosensor electroquímico basado en oligonucleótidos (aptámeros) para la detección de trombina sin el previo marcaje de este analito, ni la adición de reactivos para la detección del analito. En el sistema más sensible se obtuvo un límite de detección de 30 fM en un tiempo de respuesta de solo 5 minutos

Lupin has recently been added to the list of allergens requiring mandatory advisory labelling on foodstuffs sold in the European Union, and since December 2008 all products containing even trace amounts of lupin must be labelled correctly. Lupin globulins consist of two major globulins called α-conglutin (11S and “legumin-like”) and β-conglutin (7S and “vicilin-like”), and another additional two globulins, γ-conglutin and δ-conglutin, which are present in lower amounts. β-conglutin is the only conglutin currently included in the list of the International Union of Immunological Societies (IUIS), designated as Lup an 1. The overall objective of these PhD is the selection of aptamers that can detect this allergen. Nucleic acid aptamers are synthetic ligands selected from vast combinatorial libraries through a process referred to as SELEX – Systematic Evolution of Ligand By Exponential Enrichment. Aptamers possess unique chemical and biochemical characteristics, such as: well known chemistry and remarkable stability, moreover, aptamers can be selected against virtually any target and in non-physiological conditions. In order to achieve the overall objective, a set of subobjectives will be achieved. The first of these involves the elucidation of protocols for the selective extraction of each of the lupin α, β, γ, and δ subunits, resulting in (i) protocols that can be used for selective extraction and isolation of the lupin α, β, γ, and δ proteins from food for subsequent analysis; (ii) standards that can be used in analytical assays and tools; and (iii) target that can be used for the selection of aptamers specific to the β-conglutin subunit. The core of the work is the selection of aptamers against the allergen Lup an 1 using a SELEX procedure, as well the preparation of protocols that can be used to monitor the evolution of aptamer selection. The functionality of the aptamer is demonstratedby exploiting it in an enzyme linked oligonucleotide assay as well as apta-PCR. Finally the resulting aptamer candidates that exhibit high affinity are fully characterised, truncated, and the structure of the final truncated aptamer is elucidated

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2014.
Cataloged from PDF version of thesis. Vita.
Includes bibliographical references.
The structural dynamics of nucleic acids are intimately related to their biological functions; however, our ability to study these molecular dynamics has been largely impeded by the lack of techniques that possess both high time resolution and structural sensitivity. The motivation for the work in this thesis was to develop and apply two-dimensional infrared spectroscopy (2D IR) as a new experimental tool to investigate nucleic acid dynamics. Infrared spectroscopy is sensitive to structural changes of nucleic acids and 2D IR offers sub-picosecond time resolution. 2D IR spectroscopy is advantageous over the linear infrared absorption spectroscopy because the vibrational spectrum is spread onto two frequency axes, giving rise to the structurally sensitive cross-peaks. These cross-peaks allow the determination of vibrational couplings, which encode chemical bond connectivity, distance and orientation. However, 2D IR spectroscopy of nucleic acids is underdeveloped due to the difficulties in modeling highly delocalized and coupled vibrations of nucleobases. This thesis initiated the efforts to develop 2D IR spectroscopy of nucleic acids by first characterizing the 2D IR spectra and vibrational eigenstates of nucleobases, using a model of multiple anharmonically coupled oscillators. With pronounced cross-peaks existing between all the vibrations for a give nucleobase, 2D IR spectroscopy was shown to be capable of distinguishing between different tautomers, using pyridone as a model system. Coupled with a laser-induced temperature-jump (T-jump), 2D IR was used to monitor rapidly exchanging tautomers in real time under physiological conditions on the nanosecond timescale. Systematically characterizing the tautomer exchange rates as a function of various experimental variables lead to a two-state concerted mechanism involving bridging water wires for the lactam-lactim tautomerization of 6-chloro-2-pyridone. This method was then applied to study the tautomerism of a deoxycytidine analog, KP1212, which is an anti-HIV drug. Multiple tautomers, including the normally rare enol tautomers, were found under physiological conditions. This observation supports the rare tautomer hypothesis, which states that each tautomer displays a distinct base-pairing preference, eventually leading to mutations and population collapse of the HIV viruses. Beyond studies on the single nucleotide level, 2D IR was used to characterize the structural dynamics of thrombin-binding aptamer (TBA), which is a 15mer DNA folded into a guanine-quadruplex (G-quadruplex). The 2D IR spectral signatures of G-quadruplex were established, and T-jump transient 2D IR was employed to investigate the unfolding dynamics of TBA. A mechanism of the early unfolding of TBA was proposed: A ~100 nanosecond response was attributed to the local deformation of the G-quadruplex, and a few-microsecond response was ascribed to be the fraying of the 3'-tail of TBA. This observation was consistent with a mechanism suggested by molecular dynamics simulations. Finally, the dissociation of double-stranded DNA formed by TBA and its complementary strand was found to be on the timescale of tens to hundreds of microseconds. The experiments in this thesis demonstrate the capability of 2D IR to investigate nucleic acid dynamics spanning a wide range of timescales.
by Chunte Sam Peng.
Ph. D.