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No. of bitstreams: 2 Dissertação - Rívia Aparecida Reinalda Arruda - 2016.pdf: 3000238 bytes, checksum: 221f622157672c4f34ee32f2aed12d0b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-09-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The main purpose of this study was to assess conformational changes in preselected aptamers A03 and C10 against untranslated regions present in the genome of the dengue virus (DENV), by means of molecular biology and physics assays. Aptamers are nucleic acid molecules with potential theranostic applications. In this context, dengue was the target chosen for study because is an important infectious viral disease presenting high rates of morbidity and mortality in the worldwide, especially in tropical and subtropical areas, where there are high incidences of the mosquito vector Aedes aegypti. These aptamers showed, by dot-blot and magnetic beads ligand capture and subsequent qPCR detection, similar affinities and specificities to binding to the DENV 5'UTR. Probably, the analogy of the motifs and secondary structures presented in these ligands, identified by in silico studies, are responsible for these similarities. Data obtained by using Fluorescence Resonance Energy Transfer Assays (FRET) confirmed the affinity of A03 as DENV ligand by conformational changes in the double labeled aptamer (5’-FAM, 3’-TAMRA). Aptamer conformacional changes resulting from its interaction with the target were detected by the modifications in the fluorescence intensities between FAM and TAMRA fluorophores. These conformational changes results in donor-acceptor distance modification that has an effect on FRET efficiency and on fluorescence intensity of these molecules. The ionic strength of the buffer used in the trials had significant influence on the conformation of the aptamer, allowing the detection of interactions between A03 and DENV. The ions in the buffer lead an effect in the conformation of the aptamer, increasing its thermal stability, and the Mg2+ ion has led to major conformational changes on A03 compared to Na+ ion. The data presented herein demonstrated the importance of the studies of oligonucleotides binding affinity and specificity to Dengue virus. It can be concluded that the A03 and C10 aptamers have potential in application for diagnostic and/or therapeutic purposes, considering the diluent buffer, since different salts have influence on the secondary and tertiary conformational structures of aptamers, and on bioavailability of interaction with the target. / O objetivo principal desse trabalho foi avaliar alterações conformacionais nos aptâmeros A03 e C10 previamente selecionados contra regiões não traduzidas presentes no genoma do vírus da dengue (DENV), por meio de testes de biologia e física molecular. Os aptâmeros são moléculas de ácidos nucleicos com potencial aplicação na teranóstica das mais diversas doenças. Nesse contexto, destaca-se a dengue, uma doença infecciosa viral com altos índices de morbi-mortalidade em um cenário mundial, principalmente em regiões tropicais e subtropicais, onde são altas as incidências do mosquito vetor Aedes aegypti. Esses aptâmeros demonstraram, por meio de dot blot e de captura de ligantes por beads magnéticos com detecção por PCR em Tempo Real, afinidades e especificidades semelhantes de ligação à 5’UTR do DENV. Provavelmente, essas semelhanças se deram em função dos motivos e das estruturas secundárias análogas apresentadas por esses ligantes, obtidas por ensaios in silico. Os ensaios de Transferência Ressonante de Energia de Fluorescência (FRET) permitiram a confirmação dessa afinidade de ligação entre A03:DENV, devido às alterações estruturais ocorridas no aptâmero duplamente marcado em sua sequência (5’-FAM; 3’-TAMRA). As alterações conformacionais no aptâmero resultantes de sua interação com o alvo foram detectadas pelas mudanças nas intensidades de fluorescência do FAM e do TAMRA, decorrentes de alterações nas distâncias entre o doador e o aceitador que afetam a eficiência FRET e por conseguinte as intensidades de fluorescência destas moléculas. A força iônica do tampão utilizado nos ensaios apresentou importante influência na conformação do aptâmero, permitindo a detecção das interações entre A03 e o DENV. Os íons presentes na solução tampão afetaram a conformação estrutural do aptâmero e aumentaram sua estabilidade térmica, sendo que o íon Mg2+ levou a maiores alterações estruturais sobre o A03 comparado ao íon Na+. Os dados aqui apresentados demostraram a importância dos estudos de afinidade e especificidade dos oligonucleotídeos ligantes ao vírus da dengue. É possível concluir que os aptâmeros A03 e C10 apresentam potencial de aplicação para fins diagnósticos e/ou terapêuticos, considerando o tampão diluente, uma vez que os diferentes sais presentes exercem influência sobre as estruturas conformacionais secundárias e terciárias dos aptâmeros, e em sua biodisponibilidade de interação com o alvo.

An ideal biosensor is a compact and in-expensive device that is able to readily and rapidly detects different types of analytes with high sensitivity and specificity. The affectability of a biosensing methodology is subject to the limit of nanomaterials to transduce the target binding process to an improved perceptible signal, while the selectivity is accomplished by considering the binding and specificity of certain moieties to their targets. Keeping these requirements in mind we have chosen nanomaterials such as carbon nanotubes (CNTs) and gold nanoparticles (AuNPs) that has catalytic properties combined with their size, shape and configuration dependent chemical and physical properties as essential precursors and signaling components for creation of biosensors with tremendous sensitivity. The primary goal of the research work described in this dissertation is to develop and evaluate novel methods to detect various analytes using nanomaterials, at the same time making an affordable architecture for point-of-care (POC) applications. We report here in chapter 3 a simple and new strategy for preparing disposable, paper-based, porous AuNP/M-SWCNT hybrid thin gold films with high conductivity, rapid electron transfer rates, and excellent electrocatalytic properties to achieve multiple analyte electrochemical detection with a resolution that greatly exceeds that of purchased flat gold slides. We further explored the use of nanomaterial-based paper films in more complex matrices to detect analytes such as NADH, which can act as a biomarker for certain cellular redox imbalances and disease conditions. Carbon nanotubes with their large activated surfaces and edge-plane sites (defects) that are ideal for performing NADH oxidation at low potentials without any help of redox mediators minimizing surface fouling in complex matrices is described in chapter 4. With an instrument-free approach in mind we further focused on a colorimetric platform using split cocaine aptamers and gold nanoparticles (AuNPs) to detect cocaine for on-site applications as described in chapter 5. In chapter 5, the split aptamer sequences were evaluated mainly on three basic criteria, the hybridization efficiency, specificity towards the analyte (cocaine), and the reaction time to observe a distinguishable color change from red to blue. The assay is an enzyme-assisted target recycling (EATR) strategy following the principle that nuclease enzyme recognizes probe–target complexes, cleaving only the probe strand releasing the target for recycling. We have also studied the effect of the number of binding domains with variable chain lengths on either side of the apurinic (AP) site. On the basis of our results, we finally shortlisted the sequence combination with maximum signal enhancement fold which is instrumental in development of colorimetric platform with faster, and specific reaction to observe a distinctive color change in the presence of cocaine.

Le développement d’outils de détection in situ (IDT) est indispensable pour rapporter entemps réel la présence d’une signature moléculaire spécifique. L’efficacité d’un IDT estliée à son affinité, à sa spécificité, mais aussi à son potentiel à opérer dans des conditionsimposées par le contexte d’application. Parmi les contraintes imposées, la variation detempérature, le pH et la présence de solvant d’extraction. Le but du projet est dedévelopper des aptamères capables d’opérer en présence de solvant organique ; à ceteffet, nous avons opté pour une sélection en présence de méthanol. La limitation decette stratégie est principalement liée à la nature chimique des acides nucléiques. Parconséquent, nous avons choisi d’utiliser une banque ADN incorporant le (5-(octa1,7-diynyl)-2’-deoxyuridine) dDOTP, au lieu du dTTP. Notre stratégie expérimentale aabouti à la sélection d’un aptamère qui lie spécifiquement l’adénine en présence de 25 %de méthanol. Nous avons montré que les dDOTP sont essentiels à l’interaction del’aptamère avec l’adénine ; l’aptamère sélectionné comporte un motif riche en G partagéavec l’aptamère anti-adénosine, cependant, l’aptamère sélectionné dans le méthanolcomporte un motif structural indispensable à l’interaction avec la cible en présence deméthanol. Par ailleurs, nous avons montré que l’aptamère sélectionné pourraitfonctionner comme un module de reconnaissance spécifique d’un biocapteur. / Development of in situ detection tools (IDT) is required for specific real time monitoringof chemical species. Efficient IDT is not only related to its affinity and ability todiscriminate between molecular variants, but moreover it must be adapted for operatingunder conditions imposed by the context of application. Among others, hightemperature, pH variation and presence of organic solvents may be mentioned. The aimof our project is to develop an aptamer operating in the presence of organic solvents. Forthis purpose, we opted for selection in presence of methanol. Limitations of this strategyare adaptability of SELEX technology and chemical diversity of nucleic acids. For thispurpose, we used library incorporating (5-(octa1,7-diynyl)-2’-deoxyuridine) dDOTPinstead of conventional thymine nucleotide. Our experimental strategy led to theselection of an aptamer that specifically recognises adenine in the presence of 25 % ofmethanol. dDOTP nucleotides are essential for adenine recognition; the selectedaptamer shares a purine rich motif with a previously described adenosine aptamer, butdisplays a specific structure motif, essential for operating in the presence of methanol.Furthermore, the ability of truncated variants of the selected aptamer to form arecognition module of biosensor was assessed in this study.

Des approches thérapeutiques visant à la stimulation de la régénération et/ou à l’inhibition des processus de dégénérescence neuromusculaire pourraient constituer des stratégies efficaces pour préserver le tonus musculaire des patients et augmenter ainsi leur espérance de vie. L’activation de la Calcineurine A (CnA), une phosphatase des sérines et thréonines, contrôle une large gamme de réseaux régulateurs dans le muscle squelettique, notamment en stimulant l’expression de gènes spécifiques des fibres musculaires lentes (de type I). La CnA est considérée comme un acteur clé de la réponse hypertrophique et du processus de régénération dans le muscle squelettique. L’activation de la CnA est ainsi considérée comme une stratégie potentielle pour stimuler la régénération musculaire dans les cas de myopathie. Nous avons identifié un aptamère peptidique qui active la CNA in vitro et in vivo. Dans un modèle murin d’atrophie musculaire induite par dénervation, l’aptamère a montré de significatives capacités thérapeutiques. L’effet curatif de l’aptamère a notamment été observable par une augmentation générale de la surface des muscles traités, mais aussi par un accroissement de la surface individuelle des fibres musculaires.Une augmentation du niveau de NFAT nucléaire dans ces fibres a été observée, en cohérence avec les capacités d’activation de la CnA par notre aptamère. Par ailleurs, une autre observation faite dans les muscles traités avec l’aptamère a été l’augmentation de noyaux centraux, caractéristiques de la présence de nouvelles fibres. Finalement, l’identification du site d’interaction entre la CnA et notre aptamère, permise par l’utilisation de plusieurs formes tronquées de la phosphatase, a offert un aperçu du mécanisme d’action de l’aptamère à l’échelle moléculaire. Dans l’ensemble, les études présentées ici ont offert la première démonstration qu’une activation directe de la CnA endogène a un impact significatif sur les processus cellulaires, résultant en la stimulation de la régénération musculaire et l’amélioration de l’état physiopathologique chez les modèles animaux utilisés. / Therapeutic approaches leading to the stimulation of regeneration, and/or inhibition of degeneration processes in neuromuscular disorders are believed to offer valid therapeutic strategies that would preserve muscle tone and contribute to the quality of life while lengthening patient life span. Activation of CalcineurinA (CnA), a threonine-serine phosphatase, controls gene regulatory programs in skeletal muscle by stimulating slow muscle fiber (type I) gene expression. This phosphatase has been also identified as a key mediator in the hypertrophic response and in skeletal muscle regeneration. Activation of CnA is, therefore, considered as a potentially interesting means of stimulating muscle regeneration in myopathies. We have identified a peptide aptamer that activates CnA in vitro, in cells and in vivo. In a mouse model for denervation-induced muscle atrophy, CnA-activating peptide aptamers show significant positive impact. This is reflected in larger overall muscle cross-sectional surface area due to an increased number of fibers and larger individual fiber surface area. Insight into the biological mechanism is afforded by observation of increased levels of nuclear NFAT transcription factor in these fibers, in agreement with peptide aptamer-mediated activation of CnA. Furthermore, a significant increase in central nuclei, characteristic of the presence of new fibers, is observed in muscles treated with the peptide aptamers specifically activating CnA. Identification of the specific binding site of the peptide aptamer on CnA was achieved using several truncations of the phosphatase, offering insight into the molecular mechanism of action. Together, these studies offer the first proof that direct activation of endogenous CnA has a measureable impact on cellular responses resulting in stimulation of muscle regeneration and enhancement of pathophysiological state in selected animal models. / Specifici approcci terapeutici diretti alla stimolazione della rigenerazione e/o dell’inibizione dei processi degenerativi in patologie neuromuscolari, sono considerati come strategie efficaci per preservare il tono muscolare e aumentare in questo modo la speranza di vita dei pazienti. L’attivazione della Calcineurin A (CnA), una treonina/serina fosfatasi, controlla una vasta gamma di vie di trasduzione nel muscolo scheletrico, stimolando in particolare l’espressione dei geni specifici delle fibre muscolari lente (tipo 1). La Cna rappresenta un elemento chiave nella risposta ipertrofica e nel processo di rigenerazione muscolare. Per questo motivo, l’attivazione della CnA é considerata come un’approccio terapeutico interessante per stimolare la rigenerazione muscolare nelle miopatie. Nel nostro laboratorio, abbiamo identificato un aptamero peptidico che attiva la CnA sia in vitro che in vivo. In un modello murino di atrofia muscolare indotta tramite denervazione, l’aptamero petidico risulta avere delle significative potenzialità terapeutiche. Tale effetto si riflette in un aumento della superficie totale delle sezioni trasversali dei muscoli trattati, dovuto all’aumento sia del numero delle fibre che alla loro superficie individuale. L’effetto dell’aptamero peptidico sull’attivazione della CnA , nelle fibre trattate in vivo é dimostrata dall’osservazione della localizzazione prevalentemente nucleare del fattore di trascrizione NFAT, principale substrato della CnA. Un notevole aumento di nuclei centrali, caratteristica principale del processo di rigenerazione muscolare, é inoltre osservato in queste fibre. L’identificazione del sito d’interazione dell’aptamero peptidico e la proteina tramite l’utilizzo di vari costrutti della CnA ha permesso di avanzare delle ipotesi sul meccanismo d’azione dell’aptamero a livello molecolare. In conclusione, gli studi esposti in questa tesi rappresentano la prima dimostrazione che la diretta attivazione della CnA endogena ha un notevole effetto sulla stimolazione della rigenerazione muscolare e porta al miglioramento dello stato fisio-patologico nei modelli murini utilizzati.

A new detection reagent that could possibly augment or replace antibodies research and diagnosis methods are aptamers. Aptamers are ssDNA, RNA or polypeptide constructs that function like active antibodies. Antibodies and aptamers both specifically bind to selected target molecules, and as such they enable the detection or targeting of the presence or absence of a specific antigen. In order to ensure that ssDNA aptamers perform similarly to antibodies, anti-VEGF-A polyclonal antibody and anti-VEGF-A ssDNA aptamer were evaluated against vascular endothelial growth factor A (VEGF-A) using Surface Plasmon Resonance (SPR). It was hypothesized that the anti-VEGF-A aptamer had the same, if not better, binding kinetics than the anti-VEGF-A polyclonal antibody, and as such offers an ideal replacement for use in in field, real-time testing assays. SPR revealed that both the polyclonal antibody and ssDNA aptamer bound the target antigen, VEGF-A. Additionally, from the SPR kinetic analysis, the anti-VEGF-A aptamer had KD values of 20-28 nM and the anti-VEGF-A antibody had KD values of 16-127 uM. The binding efficacy of the aptamer was several orders of magnitude better than that of the antibody. The aptamer was also stable in solution for a longer amount of time than the antibody, which denatured in solution after two weeks.

The purpose of this thesis was to demonstrate device functionality of 3-D paper-based, multiplex platforms, µPADs, without the use of coupling agents between layers. Previously, these platforms were fabricated with double-sided tape and cellulose powder to try to augment proper fluid routing, but difficulties with this method occurred. An acrylic housing unit with strategically placed pressure tabs was designed to aid horizontal and vertical fluid routing through the platform, thus eliminating the inconsistencies associated with coupling agents. Channel characterization studies, a COMSOLTM simulation, and development time studies were performed to aid device design and demonstrate device functionality. The implementation of this µPAD platform as a diagnostic instrument was validated via lateral flow immunoassays utilizing both biotinylated antibodies and biotinylated aptamers as capture reagents. Successful detection of the target analyte, IgE, as well as successful fluid routing through multiple layers of membrane was demonstrated by immunoassays performed on 3-D, multiplex platforms. Another important result determined the aptamers’ ability to detect IgE to be statistically the same as the antibodies’ ability; thus confirming aptamers as viable capture reagent alternatives to antibodies in lateral flow assays. Overall, this research project was performed to develop and validate via experiment a prototype paper-based microfluidic diagnostic device, µPAD, with the capability to detect multiple biomarkers on one platform.

<p>The process of ‘‘structure-switching’’ enables biomolecular switches to function as effective biosensing tools. Biomolecular switches can be activated or inactivated by binding to a specific target that triggers a precise conformational change in the biomolecules involved. Examples of aptamer-based biomolecular switches can be found in nature. Furthermore, efforts have been made in the last decade to engineer structure-switching sensors using DNA aptamers whereby, the aptamer is coupled to a signal transduction method to generate a readout signal upon target binding to the aptamer domain. Conversely, RNA aptamers have been relatively underexplored for sensor development, largely due to its susceptibility to nuclease degradation and chemical instability. Despite these shortcomings, many RNA aptamers possess superior sensing capabilities, and the abundance of RNA aptamers provides new opportunities to further advance the field. In effect, this thesis uses a structure-switching design to demonstrate the power of RNA aptamers for fluorescence-based sensor development. Herein, we demonstrate generalizable structure-switching strategies to make use of the abundance of RNA aptamers, monitor the quality control of detection and correct detection error, as well as enhance RNA aptamer sensing capability by using regulated graphene adsorption. Furthermore, our findings have expanded for secondary applications involving collaborations with other research labs. In one application, we demonstrate that entrapment of structure-switching RNA aptamers in sol-gel material confers protection against nuclease degradation and chemical instability. In another application, we further validate the use of riboswitches, or natural structure-switching RNA aptamers, as potential targets for drug discovery. Overall, these results demonstrate the capability of RNA aptamers for sensor development. We conclude with a discussion of possible areas for further inquiry, as well as future applications for the advancement of structure-switching RNA aptamers.</p> / Doctor of Philosophy (PhD)

京都大学 / 新制・課程博士 / 博士(エネルギー科学) / 甲第25395号 / エネ博第474号 / 新制||エネ||89(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 片平 正人, 教授 森井 孝, 教授 杤尾 豪人 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

During its intraerythrocytic stages (IE), Plasmodium falciparum, the causative agent of the deadliest human malaria, remodels the host red cell membrane with a poorly defined assortment of parasite-­encoded proteins that undergo antigenic variation. Despite the requirement for immunologic stealth, exported parasite proteins also mediate strain-independent functions such as endothelial sequestration that are critical for parasite survival and pathogenesis. This thesis explores the hypothesis that P. falciparum displays novel structurally conserved proteins on the IE surface and these proteins may serve as useful antigens for a broadly effective anti-­malarial vaccine. In order to test this hypothesis, we developed an in vitro selection technique that sequentially incorporates unique P. falciparum isolates as the targets for Systematic Evolution of Ligands by EXponential enrichment (Serial-SELEX) to generate nucleic acid molecular probes, aptamers, capable of recognizing conserved cell surface determinants. Ten of 11 enriched aptamers were -parasite selective and three of these aptamers demonstrated strain-independent binding to P. falciparum. Aptamer recognition extended beyond the parasites used in Serial-SELEX to other laboratory and recent field isolates. Surprisingly the same three broadly binding aptamer selected against P. falciparum also recognized all laboratory-adapted and clinical isolates of P. vivax and P. knowlesi tested, strongly supporting our hypothesis that structurally conserved molecules are present on the surface IEs. Competition studies showed that the aptamers bound a single target which was confirmed as an IE membrane protein. Aptamer­‐mediated affinity purification and tandem mass spectrometry enabled identification of the aptamer target as parasite-encoded protein. Discovery of a protein conserved between the major human malarias may have implications for vaccine development and validates the Serial‑SELEX technique as a powerful tool for antigen discovery.

L'une des stratégies mise en œuvre pour améliorer la prise en charge thérapeutique des patients concerne le développement d'outils diagnostiques sensibles et spécifiques. Les aptamères sont des oligonucléotides artificiels obtenus par SELEX avec une très haute affinité ainsi qu'une excellente spécificité pour leurs cibles. L'immobilisation de ces motifs de reconnaissance moléculaire à la surface de nanomatériaux tels que des nanoparticules d'or (AuNPs), dont les propriétés optiques et électroniques sont uniques, permet d'amplifier le signal généré par l'interaction du ligand avec sa cible. Deux systèmes de biosensing ont été élaboré en fonctionnalisant des AuNPs avec des aptamères, l'un dirigé contre la thrombine et le second dirigé contre une marque épigénétique portée par une protéine histone. La réduction des sels d'or aurique précurseurs a été réalisée en présence de PEG4 et a conduit à l'obtention d'une population homodisperse de AuNPs sphériques d'un diamètre moyen de 14 nm et présentant une isotropie de taille et de forme. Ces AuNPs ont ensuite été fonctionnalisées par des bras espaceurs de longueur variable constitués d'unités tétraéthylène glycol successives reliées entre elles par des ponts éthers ou triazoles. L'acide lipoique a été utilisé comme motif d'ancrage à la surface des AuNPs via une liaison covalente Au-S et a été couplé aux différents bras espaceurs via une réaction de Steglich. Les linkers étaient porteurs d'un groupement terminal azoture afin de réaliser le couplage par chimie-click avec les aptamères. La stratégie de détection de la thrombine utilisait les propriétés de quenching de fluorescence des AuNPs alors que la détection de l'histone était colorimétrique et mettait à profit l'effet de résonance plasmonique de surface des nanoparticules d'or. / Improving patients therapeutic care needs the development of sensitive and specific diagnostic tools. Aptamers are synthetic oligonucleotides obtained by SELEX with a very high affinity and excellent specificity for their targets. Grafting of these molecular recognition patterns onto nanomaterials such as gold nanoparticles (GNPs), which unique optical and electronic properties, can amplify the signal induce by the interaction between the ligand and its target. Two biosensing systems have been developed by GNP functionalization with aptamers, one is directed against thrombin and the second against an epigenetic mark carried by a histone protein. Gold precursors was reduced in the presence of PEO4 and led to a homodisperse population of spherical GNP with an average diameter of 14 nm and an isotropy of size and shape. GNP were functionalized with tetraethylene glycol units interconnected by ether or triazoles bridges as a linker. Lipoic acid was used as an anchor moiety onto gold surface via a covalent Au-S bond and was coupled to the spacer through a Steglich reaction. The linkers were functionalized with an azide group to perform the coupling with aptamers by click chemistry. The thrombin sensing strategy used the fluorescence quenching properties of GNPs while the histone detection involved the gold nanoparticle plasmon resonance surface effect.