Actualmente, el antígeno prostático específico (PSA) se considera el marcador más sensible para la detección de cáncer de próstata. Hasta la fecha anticuerpos monoclonales y policlonales se han utilizado para detectar el PSA debido a su alta especificidad y sensibilidad. Sin embargo, la producción de anticuerpos es lenta y cara. En el otro lado, los aptámeros con especificidad y afinidad iguales a los de los anticuerpos podría ser una forma alternativa para la detección de PSA. El objetivo de este trabajo es la selección de aptámeros contra el PSA. Aspectos fundamentales como la caracterización de la PSA, la evaluación de las estrategias de inmovilización, la preparación de la biblioteca de oligonucleótidos y de la cadena simple de ADN (ssDNA) han sido evaluados con el fin de alcanzar el objetivo principal de esta tesis. Finalmente, la selección de aptámeros contra el PSA y su uso potencial en aplicaciones de diagnóstica y terapéutica han sido descritos. / Currently, PSA is considered the most sensitive marker available for prostate cancer detection and for monitoring its progression. To date monoclonal/polyclonal antibodies have been used to detect PSA due to their high specificity and sensitivity. However, the production of antibodies is time-consuming and expensive. On the other hand aptamers with specificity and affinity equal to those of antibodies could be an alternative way for the detection of PSA. This work overviews the selection of aptamers against prostate specific antigen (PSA). Fundamental aspects such as the characterization of PSA, evaluation of immobilization strategies, the preparation of oligonucleotide library and single stranded DNA (ssDNA) have been evaluated in order to reach the main objective of this thesis. Finally, selection of DNA and RNA based aptamers against PSA and their potential use in diagnostic and therapeutic applications have been described

SELEX is a selective amplification technique based on the assumption that functional nucleic acids (FNAs) can be found in a pool of chemically synthesized random nucleic acid sequences. These FNAs can either bind to a target of interest (aptamers) or catalyze a chemical reaction (DNAzymes and ribozymes) or both (aptazyme). The aptazymes discussed herein, are RNA-cleaving DNAzymes that become catalytically active upon target recognition at the aptamer domain. In chapter 2, two SELEX experiments were performed in parallel to determine whether it was possible to obtain multiple aptazymes from a single SELEX procedure. Two different crude intercellular mixtures (CIM) each containing 35 unique overexpressed ASKA clone gene products were used. After 10 rounds of SELEX, the sequence pool was analyzed using Illumina Genome Analyzer. The sequences tested appeared to bind to a protein other than the 35 gene products of interest. To redirect these SELEX experiments towards the gene products of interest, 12 gene products from each parallel selection was purified and incubated with sequences from round 6 of the CIM SELEX. A total of 13 rounds of selection were performed using these purified proteins. The sequences were analyzed and found to either not cleave at all or self-cleave. In chapter 3 a similar SELEX approach was explored using 36 purified proteins, from the CIM SELEX to isolate protein-binding DNA aptamers. This SELEX approach served as another means to explore isolating multiple aptamers against multiple targets. However both the RNA-cleaving aptazyme and protein-binding DNA aptamer SELEX experiments both experienced challenges in non-specific binding. Although negative selection steps were taken in order to avoid non-specific binding species, such sequences were still isolated. In order for these approaches to be successful, negative selection steps are required to remove any self-cleaving and non-specific FNAs. Although these studies did not conclusively give rise to the desired FNAs, it has produced some insight to the potential setbacks associated with developing a screen for multiple targets. / Thesis / Master of Science (MSc)

RNA aptamers that bind to a wide range of targets with high affinity and specificity have been identified via the in vitro systematic evolution of ligands by exponential enrichment (SELEX). However, the process is quite unpredictable due in part to binding that occurs not only on the targets themselves but also on any of the other functional groups, moieties, or surfaces. Recent modelling work has shown that this level of “background binding” is a key parameter in the performance of aptamer selection processes. One strategy to minimize the amount of background binding is to pre-block those possible binding sites with a non-amplifiable nucleic acid molecule, such as yeast tRNA. It is also known that binding buffer conditions have strong effect on the binding affinity of nucleic acids. However, there are no detailed studies and little quantitative information available to guide the design of aptamer selection processes. In this study, the binding ability of yeast tRNA, which has comparable size with most RNA aptamer libraries, on both silicon dioxide and poly (ethylene terephthalate glycol) (PET-G) surfaces was studied using Quartz Crystal Microbalance with Dissipation (QCM-D). Silicon dioxide surface is a commonly used substrate for QCM-D tests on the adsorption behaviour of different nucleic acid. PET-G is a commonly used polymer substrate for the fabrication of microfluidic devices, which are advanced techniques for aptamer selection. The presence of specific divalent cations, for example Mg2+ over Ca2+, in binding buffers greatly enhanced the binding of yeast tRNA on silicon dioxide surfaces and PET-G surfaces. Proper NaCl concentration (100 mM) and MgCl2 concentration (5 mM) is necessary to enhance yeast tRNA binding on both surfaces. Yeast tRNA binding ability on silicon dioxide surfaces show more dependence on binding buffer pH than on PET-G surfaces. / Thesis / Master of Applied Science (MASc)

The focus of this work was the development and application of highly efficient RNA catalysts for the site-specific modification of RNA with special focus on methylation. In the course of this thesis, the first methyltransferase ribozyme (MTR1), which uses m6G as the methyl group donor was developed and further characterized. The RNA product was identified as the natural modification m1A. X-Ray crystallography was used to solve the 3D structure of the ribozyme, which directly suggested a plausible reaction meachnism. The MTR1 ribozyme was also successfully repurposed for a nucleobase transformation reaction of a purine nucleoside. This resulted in a formyl-imidazole moiety directly on the intact RNA, which was directly used for further bioconjugation reactions. Finally, additional selections and reselections led to the identification of highly active alkyltransferase ribozymes that can be used for the labeling of various RNA targets / Der Schwerpunkt dieser Arbeit lag auf der Entwicklung sowie Anwendung hocheffizienter RNA-Katalysatoren für die positionsspezifische Modifikation von RNA mit besonderem Fokus auf Methylierungen. Im Rahmen dieser Arbeit wurde das erste Methyltransferase-Ribozym (MTR1), das m6G als Methylgruppendonor verwendet, entwickelt und näher charakterisiert. Das RNA-Produkt wurde als die natürliche Modifikation m1A identifiziert. Mit Hilfe der Röntgenkristallographie wurde des Weiteren die 3D-Struktur des Ribozyms aufgeklärt, was direkt auf ein plausibles Reaktionsmuster schließen ließ. Das MTR1-Ribozym wurde zudem erfolgreich für eine Nukleobasen-Transformationsreaktion eines Purins verwendet, bei der eine Formyl-Imidazol-Einheit direkt an der intakten RNA gebildet wird. Dieses Reaktionsprodukt wurde für positionsgenaue Biokonjugationsreaktionen verwendet. Schließlich führten zusätzliche Selektionen und weitere Reselektionen zur Identifizierung hochaktiver Alkyltransferase-Ribozyme, die für die Markierung verschiedener Ziel-RNAs verwendet werden können.

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.

Philosophiae Doctor - PhD / Breast cancer is the second most common cancer amongst South African women. Despite ongoing efforts to combat breast cancer, current prognostic and/or therapeutic monitoring methods are limited since very little improvement, in the rate of long term recurrence of breast cancer, has been observed. Considering this, developing novel strategies to detect breast cancer recurrence – at an early onset – is crucial for monitoring the disease and potentially preventing disease progression. Methods currently used for the detection of BC are costly and can also be very uncomfortable for the patient. These methods are also too costly to use as a routine test, following surgery or treatment to assess disease progression. Thus, developing a cost-effective detection method appears to be an appealing alternative. Serum/blood-based biomarkers are ideal targets for the development of low cost detection assays. Two candidate biomarkers, unique ligand binding protein 2 (ULBP2) and glial cell line-derived neurotrophic factor family receptor alpha 1 (GFR1) were identified using bioinformatics and proteomics, respectively. These biomarkers have demonstrated to be useful prognostic biomarkers for breast cancer. The selection of aptamers against these biomarkers can facilitate the development of cost-effective detection methods. Aptamers are short DNA or RNA oligonucleotides that have very high affinity and specificity for its targets and can potentially replace antibodies as tools for molecular recognition in detection systems, such as the enzyme-linked immunosorbent assay (ELISA), lateral flow assays and electrochemical biosensors.

Thesis advisor: Michelle M. Meyer / Thesis advisor: Peter Clote / Structured RNAs have many biological functions ranging from catalysis of chemical reactions to gene regulation. Many of these homologous structured RNAs display most of their conservation at the secondary or tertiary structure level. As a result, strategies for natural structured RNA discovery rely heavily on identification of sequences sharing a common stable secondary structure. However, correctly identifying the functional elements of the structure continues to be challenging. In addition to studying natural RNAs, we improve our ability to distinguish functional elements by studying sequences derived from in vitro selection experiments to select structured RNAs that bind specific proteins. In this thesis, we seek to improve methods for distinguishing functional RNA structures from arbitrarily predicted structures in sequencing data. To do so, we developed novel algorithms that prioritize the structural properties of the RNA that are under selection. In order to identify natural structured ncRNAs, we bring concepts from evolutionary biology to bear on the de novo RNA discovery process. Since there is selective pressure to maintain the structure, we apply molecular evolution concepts such as neutrality to identify functional RNA structures. We hypothesize that alignments corresponding to structured RNAs should consist of neutral sequences. During the course of this work, we developed a novel measure of neutrality, the structure ensemble neutrality (SEN), which calculates neutrality by averaging the magnitude of structure retained over all single point mutations to a given sequence. In order to analyze in vitro selection data for RNA-protein binding motifs, we developed a novel framework that identifies enriched substructures in the sequence pool. Our method accounts for both sequence and structure components by abstracting the overall secondary structure into smaller substructures composed of a single base-pair stack. Unlike many current tools, our algorithm is designed to deal with the large data sets coming from high-throughput sequencing. In conclusion, our algorithms have similar performance to existing programs. However, unlike previous methods, our algorithms are designed to leverage the evolutionary selective pressures in order to emphasize functional structure conservation. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

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.

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

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