Vários estudos têm apontado a sinalização e os receptores purinérgicos, representados em mamíferos pelos receptores ionotrópicos (P2X1 P2X7) e metabotrópicos (P2Y1,2,4,6, 11,12,14), como um sistema primitivo, envolvido não somente na sinalização neuronal, mas também em muitos outros processos vitais incluindo resposta imune, inflamação, dor, agregação plaquetária e nos processos de diferenciação, proliferação e morte celular, que ocorrem no desenvolvimento e na regeneração tecidual. Condizente com as descrições da literatura, dados do nosso laboratório, baseados na farmacologia dos receptores purinérgicos, sugeriram o envolvimento do subtipo P2Y2 na proliferação e na neurogênese in vitro de células de carcinoma embrionário P19. Tendo em vista a ausência de agonistas e antagonistas específicos para a maioria dos subtipos de receptores purinérgicos, o que vem dificultando a elucidação das funções exatas desses receptores em processos fisiológicos e patológicos, optamos para o screening de uma biblioteca combinatória de oligonucleotídeos para a identificação de ligantes de alta afinidade e especificidade para o receptor P2Y2 (procedimento de SELEX, Evolução Sistemática de Ligantes por Enriquecimento Exponencial). Essa abordagem envolve passos reiterativos de seleção in vitro de moléculas de RNA, estabilizadas por substituição do grupo 2´OH das pirimidinas por um átomo de flúor, que possuem afinidade pelo receptor, até que a mistura de RNAs, originalmente de 1013 diferentes seqüências que adotam uma gama de estruturas secundárias e terciárias, esteja purificada para uma população homogênea de ligantes de alta afinidade pelo receptor P2Y2. O processo envolve a transcrição in vitro da biblioteca de DNA para RNA, a apresentação desta ao alvo, a eluição dos ligantes específicos, denominados aptâmeros, e a regeneração da biblioteca de DNA por RT-PCR, a qual, após uma reação de transcrição in vitro, gera a mistura de RNAs para o próximo ciclo de seleção. Neste trabalho, nós utilizamos como alvo o receptor P2Y2 recombinante humano expresso na linhagem de células de astroglioma humano 1321N1. Ao final de nove ciclos de SELEX, nós isolamos 46 sequências que foram agrupadas em três classes estruturais, de acordo com a presença de regiões consensos. A mistura destas moléculas se ligou ao receptor P2Y2 humano com uma constante de dissociação de 164 nM. Um dos clones isolados, o aptâmero B7, se ligou preferencialmente ao receptor P2Y2 (Kd 184 nM), em relação aos receptores P2Y1 e P2Y4 recombinantes expressos em células 1321N1. A interação deste aptâmero não foi dependente da espécie, uma vez que ele foi capaz de se ligar tanto ao receptor P2Y2 de origem humana como murina. A atividade biológica do aptâmero foi avaliada em células P19 (sabidamente expressando receptores P2Y2 endógenos), na qual a proteção do ATP contra a apoptose, provavelmente interagindo com o receptor P2Y2, foi anulada na presença deste aptâmero em uma concentração mil vezes menor do que a do ATP. Além de confirmar a viabilidade da técnica SELEX para identificar ligantes subtipos-específicos dos receptores purinérgicos, o aptâmero anti-P2Y2 serve como ferramenta fundamental para definir demais funções fisiológicas deste receptor. Passos de otimização das suas propriedades como ligante e biodisponibilidade tornarão este aptâmero um composto de alta relevância farmacêutica. / Many published studies have focused on purinergic signaling and receptors, represented by ionotropic (P2X1 P2X7) and metabotropic (P2Y1,2,4,6,11,12,14) subtypes as a universal system which is not only involved in neuronal signaling, but also in various other vital processes including immune response, inflammation, platelet aggregation as well as differentiation, proliferation and cell death occurring during development and tissue regeneration. In agreement with other published reports, results of our laboratory based on overlapping purinergic receptor pharmacology suggest the participation of the P2Y2 subtype in proliferation and in vitro neurogenesis of P19 embryonal carcinoma cells. In view of the lack of availability of specific agonists and antagonists for most purinergic receptors making the elucidation of exact functions of these receptors in their cellular context almost impossible, we used a combinatorial oligonucleotide library approach, denominated SELEX (Systematic Evolution of Ligands by Exponential Enrichment) for the development of high-affinity and specificity ligands for the P2Y2 receptor. This approach is based on re-iterative steps of in vitro selection of 2´-fluoro-pyrimidine-modified RNA molecules for receptor-binding affinity from an RNA pool containing 1013 different sequences and secondary and tertiary structures until this pool is purified to a homogeneous population of ligands with high affinity to the P2Y2 receptor. The combinatorial DNA library is in vitro transcribed into RNA followed by target presentation of the RNA library and elution of the target-binders, denominated aptamers, and RT-PCR amplification in order to restore the DNA library used for in vitro transcription for next SELEX cycle. Following nine SELEX cycles using 1321N1 human astroglial cells expressing recombinant human P2Y2 receptors as target, we isolated 46 aptamer sequences which, based on consensus sequence motifs, were grouped in three structural groups. The mixture of the isolated sequences bound themselves to human P2Y2 receptors with a dissociation constant (Kd) of 164 nM. One of the isolated clones, the aptamer denominated B7, bound itself to P2Y2 receptors in preference to P2Y1 and P2Y4 recombinant receptors expressed in 1321N1 cells. The binding activity of the aptamer was not limited to P2Y2 receptors of human origin, as the aptamer also interacted with mouse P2Y2 receptors. The capability of the aptamer of affecting the biological activity of P2Y2 receptors was verified in P19 cells in which ATP-induced protection against apoptosis, mediated by P2Y2 receptors, was abolished in the presence of the aptamer. In addition to providing a proof of principle for the feasibility of developing purinergic subtype-specific ligands by using the SELEX technique, the anti-P2Y aptamer provides a fundamental tool for gaining insights of physiological functions of this receptor in various cellular contexts. Moreover, steps of optimization of receptor binding properties and aptamer half-life in vivo will turn this aptamer into a compound of high pharmaceutical relevance.

The introduction of prostate-specific antigen (PSA) testing about 3 decades ago led to the possibility of early detection of prostate cancer (PCa). Although PSA testing reduced the mortality rate, it is also associated with high risk of over diagnosis in patients with and without PCa. Despite the current drawbacks, it would be a challenge to replace PSA testing entirely. Instead, there is a need to develop parallel testing of other potential biomarkers that can complement the results from PSA tests. To address alternative biomarker sensing, this thesis highlights on the development of oligonucleotide-based biosensors for the detection of different biomarkers of PCa. Using PSA as a gold standard, the first study of this dissertation investigates the use of DNA aptamers to detect PSA using electrochemical impedance spectroscopy (EIS). The study compares 6-mercapto 1-hexanol chemistry with sulfo-betaine chemistry for the development of PSA aptasensor in terms of performance and selectivity. The second study focuses on glycoprofiling in order to complement PSA quantification as an additional information for reliable PCa diagnosis. This strategy was developed in a microfluidic channel with an optical read out using chemiluminescence. This study addresses one of the major problems of cross-reactivity with lectins in glycoprofiling, which can be solved using DNA aptamers. A third study concentrates on the development of an aptasensor for Alpha-Methylacyl-CoA Racemase (AMACR). AMACR has been reported for its high specificity and sensitivity to PCa. For the fabrication of the biosensor, a new strategy using polyethylene glycol was developed by electrochemical grafting it to a polypyrrole film. Since PCa diagnosis can be improved by looking at different biomarkers, an electrochemical platform for miRNA/DNA detection using a gold nanoparticle amplification strategy was also investigated. The sensor was fabricated using peptide nucleic acids (PNA) probes on gold electrodes. The study presents non-Faradaic EIS and amperometric techniques in order to exploit the inherent charges of nucleic acids. In conclusion, this thesis wants to serve as a potential orientation for overcoming the shortcomings of the current PCa testing and contribute towards the development of oligonucleotide-based biosensors for PCa biomarker detection and hopefully enhance the diagnosis and prognosis of PCa.

The field of microfluidics is expanding into what is known as paper microfluidics. This uses a paper platform rather than materials (i.e. PDMS, PMMA) that are commonly used in microfluidics research. Current devices require an expensive manufacturing process and external sources to power the device. Such devices are not practical in low resource environments. As a consequence, it is the goal of this Thesis to develop a three-dimensional, multiplexed assay chip using nitrocellulose membranes. This device comprises of multiple layers of nitrocellulose membranes with defined fluidic channels. The multiple layers are bound together using double backed tape, and imbedded between the layers are conjugate reagents. In the detection region both antibodies and aptamers were evaluated. The fiberglass pad where conjugate reagents would be contained, were initially saturated in dye. As sample was inputted into the three-dimensional chip, the fluid path could be visualized. Without the use of the conjugate pad the chip’s four detection regions showed detection within one minute of one another. However, the addition of this fibrous pad skewed time points dramatically. The hypothesis that a three-dimensional chip could be designed to detect different biomarkers in a multi-analyte sample was satisfied. However, simultaneous detection was only possible if the conjugate pad was either neglected or, possibly, a different material was used. Additionally, current lateral flow assay technologies, another research area that paper microfluidics spawns from, use antibodies in order to capture biomarkers in sample and provide visual signal to the user. However, antibodies are sensitive to denaturation with pH and temperature, whereas aptamers can withstand much more extreme environmental conditions. A two-dimensional nitrocellulose chip was designed to compare antibodies and aptamers as capture reagents to detect VEGF, using colloidal gold as a particle to visualize detection. Both monoclonal and polyclonal anti-VEGF antibodies were used and showed no signal. On the other hand, the anti-VEGF aptamer produced a visual signal when conjugated to biotin on its 5’ end. This data was further validated by a separate project analyzing the binding kinetics of the antibody and the aptamer using Surface Plasmon Resonance. Therefore, the hypothesis that aptamers could be used as a possible capture reagent in a paper microfluidic chip for the detection of VEGF was satisfied.

Aptamers are short oligonucleotide sequences (DNA or RNA) capable of high affinity and specific binding to a molecule or a family of molecules. Aptamers are lower in cost and exhibit higher reproducibility when compared to antibodies and thus are well-suited for recognition and detection of small molecular targets such as drugs of abuse and small medical biomarkers. While aptamers have been extensively utilized for development of small molecule sensors, several limitations prevent measurements of complex or real-world samples. This dissertation describes methods, technologies, and assays that were developed with the goal of producing and/or improving aptamer-based sensors for target detection in complex samples. Aptamer engineering is detailed as an important facet of maximizing aptamer-sensor sensitivity and specificity, along with adaptation to various read-out mechanisms for improved selectivity. In chapter 3, an aptamer vii sensor for cocaine is developed based on binding between the fluorophore ATMND to the cocaine aptamer which results in quenching (i.e., ‘turn-off’) of the fluorescence of ATMND. Cocaine binding results in displacement of the ATMND and recovery of the fluorescence signal. Detection of cocaine is demonstrated with an engineered cocaine aptamer with higher affinity for cocaine, permitting over a 50-fold increase in sensitivity over other aptamer-based sensors. The method can be used in dilute biological fluids (e.g., saliva) with a single step reaction (seconds) and robust signal output. In chapter 4, a new adenosine specific aptamer is identified through rational engineering of a previously reported ATP-binding aptamer. The new adenosine aptamer is utilized to develop an electrochemical sensor for detection of adenosine in undiluted serum. The method displays 40-fold higher sensitivity in undiluted serum measurements over previously reported aptamer-based sensors for adenosine but also demonstrates specificity for adenosine over ATP, ADP and AMP that has not been previously reported. In chapter 5, a nuclease-guided truncation method is developed to yield optimal structure-switching aptamer sequences for the emergent illicit drug methylenedioxypyrovalerone (MDPV) and medical biomarkers ATP and deoxycorticosterone 21-glucoside (DOG). The method intelligently removes unessential nucleotides, producing truncated aptamer sequences with structure-switching functionality. This technique will be immediately useful for simple and low-cost development of aptamer-based electrochemical sensors.

It is well known boronic acids have its unique chemistry and related applications in organic synthesis. The boronic acid functionally group also plays very important roles in medicinal chemistry and chemical biology. For example, boronic acids have been developed as potential therapeutic agents, chemical biology tools. All these applications are directly related to the unique electronic and chemical properties of the boronic acid group. Herein, several application of boronic acids have been studied: 1) several groups of compounds were found as bacterial quorum sensing inhibitors; 2) a boronate compound was developed as a probe for detecting reactive oxygen species (ROS); and 3) boronic acid-modified aptamers can be used for glycoprotein recognition.

RNA aptamers are in vitro sequences of RNA that have a high affinity for their target ligand. They have applications in therapeutics, biosensors and molecular machines. While the practical applications of aptamers are increasing, it is important to study their structure and thermodynamics to improve the understanding of these molecular tools. The malachite green aptamer (MGA) provides a model system to study the interactions between aptamer and ligand that do not involve hydrogen bonding between ligand and receptor. While the original application of this aptamer was abandoned, study of the MGA binding pocket revealed an electronegative environment that was harnessed for catalysis. MGA binding also supported the notion that aptamers bind by adaptive binding. Adaptive binding is the ability of molecules to mold themselves around the structure of a ligand thereby incorporating it into their three-dimensional fold. To further expand our understanding of MGA binding and to clarify conflicting reports of affinities, we conducted isothermal calorimetry binding studies. The results reveal that the entropy of complex formation plays a large role in determining binding affinity and ligand specificity. This data combined with previous structural studies show that metal ions are required to stabilize the complexes with non-native ligands, whereas, the complex with the original selection target is stable at low salt and in the absence of divalent metal ions. Next, competitive binding studies using isothermal titration calorimetry were conducted with the aim of understanding the adaptive nature of RNA. The results of these studies reveal that there are limits to the adaptability of the aptamer. Binding of one type of ligand reduces the affinity of the aptamer pocket to a differently shaped ligand, even if this second ligand has a significantly higher affinity. The ability of MGA to change ligand preference based on buffer conditions, and the previously reported catalysis suggested that RNA may have a potential supporting multiple functions in the same molecule. To investigate this possibility we attempted to select an aptamer that supports both ligand binding and catalysis. By conducting both a DNA and RNA selection we hoped to add to the iv collection of DNA and RNA aptamers selected for the same target. There are currently too few of these to determine if any correlation can be made between DNA and RNA sequences that bind the same target. The target of the selection was fluorescein diacetate (FDA), which was chosen with the aim that it would allow the exploration of the inherent potential of the selected aptamer to cleave FDA to fluorescein. The RNA selection proved to be more successful and an attempt was made to characterize the binding of the aptamer to its target fluorescein diacetate. Unfortunately there were complications with the binding assays, but future work is proposed that should address the issues. In order to expand the MGA catalytic repertoire attempts were made to synthesize new ligands that could exploit the catalytic potential of the MGA binding pocket. Unfortunately these attempts were unsuccessful, however further attempts are recommended. The MGA used in this study was transcribed in vitro using T7 RNA polymerase. This process is known to add extra nucleotides to the end of the transcription product. Attempts were made to eliminate the n+1 product by introducing a ribozyme or DNAzyme. These were met with difficulties resulting in low yield, however mass spectrometry revealed that n and n+1 MGA bind to ligand. This, along with secondary structure prediction suggests that MGA n+1 behaves the same as n. Overall, the results presented here provide insights into the capabilities of RNA aptamers with respect to ligand binding and catalysis.

<p> In addition to being the natural genetic information carrier, DNA can also serve as a versatile material for construction of nanoscale objects. By using the base-pairing properties of DNA, we have been able to mass-produce nano-scale structures in a variety of different shapes, upon which patterns of other molecules can be further specified. The diversity of molecules and materials that can be attached to DNA and the capability of providing precise spatial positioning considerably enhance the attractiveness of DNA for nano-scale construction. A further challenge remains to use these DNA based structures for biomedical applications. </p><p> As proof-of-concept, a DNA-based nanodevice for multivalent thrombolytic delivery is designed, which intends to employ DNA nanostructures as carriers for the delivery of tissue plasminogen activator (tPA) and plasminogen. Universal modular adapter molecules that can simultaneously bind "down" to the DNA structures and "up" to these thrombolytic drugs are further proposed. We begin with exploring the molecular recognition properties provided by biotin-avidin and aptamer-ligand pairs, and are able to achieve site-specific display of certain protein targets along the DNA nanostructure scaffold. Yet for both of these approaches, only biotinylated or specially selected proteins can be patterned. We further propose to develop single-chain diabodies (scDb) as the adapter molecules. This scDb approach is highly modular and can be extended to assemble virtually any proteins and therapeutic molecules of interests, which at the same time will greatly enhance our molecular toolbox for nanoscale construction.</p> / Dissertation

The purpose of the study is to purify human growth hormone from the fermentation broth by affinity chromatography. For this purpose, human growth hormone specific oligonucleotide aptamers are selected among an aptamer library / selected oligonucleotides were synthesized and used as ligands. Effect of pH on ligand-human growth hormone complex formation was investigated and the highest complex formation was obtained at pH= 7.0. Human growth hormone is separated from the fermentation broth with 99.8% purity and 41% overall yield. The equilibrium data obtained was described by Langmuir type isotherm where saturation constant (q0) and affinity constant (K) are calculated as 0.338 mg hGH/&igrave / mol aptamer and 0.059 mg hGH/ml, respectively. Further, equilibriumdata obtained using aptamer affinity column was described by Langmuir type isotherm where saturation constant (q0) and affinity constant (K) are 0.027 mg hGH/&igrave / mol aptamer and 1.543 mg hGH/ml, respectively. It is possible that, selected aptamer can be used for purification of bulk amounts of recombinant human growth hormone by using aptamer affinity chromatography.

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.

A versatile AFM local oxidation lithography was developed for fabricating clean protein patterns ranging from nanometer to sub-millimeter scale on octadecyltrichlorosilane (OTS) layer of Si (100) wafer. This protein patterning method can generate bio-active protein pattern with a clean background without the need of the anti-fouling the surface or repetitive rinsing. As a model system, lysozyme protein patterns were investigated through their binding reactions with antibodies and aptamers by AFM. Polyclonal anti-lysozyme antibodies and anti-lysozyme aptamer are found to preferentially bind to the lysozyme molecules on the edge of a protein pattern before their binding to the interior ones. It was also demonstrated that the topography of the immobilized protein pattern affects the antibody binding direction. We found that the anti-lysozyme antibodies binding to the edge lysozyme molecules on the half-buried pattern started from the top but the binding on the extruded pattern started from the side because of their different spatial accessibility. In addition, after incubating lysozyme pattern with anti-lysozyme aptamer in buffer solution for enough long time, some fractal-shaped aptamer fibers with 1-6nm high and up to tens of micrometers long were formed by the self-assembling of aptamer molecules on the surface. The aptamer fibers anchor specifically on the edge of protein patterns, which originates from the biospecific recognition between the aptamer and its target protein. Once these edge-bound fibers have formed, they can serve as scaffolds for further assembly processes. We used these aptamer fibers as templates to fabricate palladium and streptavidin nanowires, which anchored on the pattern edges and never cross over or collapse over each other. The aptamer fiber scaffold potentially can lead to an effective means to fabricate and interface nanowires to existing surface patterns.