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Antithrombin (AT) is a plasma serine protease inhibitor that regulates thrombin and other activated clotting factors in the intrinsic and common pathways of coagulation. As the most abundant coagulation pathway inhibitor, AT serves to maintain balance in the coagulation system by inducing an anticoagulant effect. The importance of AT is evident in cases where deficiencies of AT lead to increased risk of venous thromboembolic disease. Since an AT antagonist would be considered a procoagulant, inactivating AT could provide a novel approach to restoring hemostasis in patients with inherited or acquired bleeding disorders. In vitro selection is a powerful tool used to screen large combinatorial oligonucleotide libraries against a target molecule or protein. By employing this technique in combination with high-throughput screening, we identified novel RNA aptamer candidates capable of binding AT with high affinity and inhibiting AT’s inactivation of its main target protease, thrombin. Kinetic characterization of the most abundant aptamer candidates showed a reduction in AT-mediated thrombin inhibition of 50-60%. The most inhibitory aptamer, R6_15, mediated a decrease of AT’s second-order rate constant from 2.37 ± 0.06 x 104 to 1.57 ± 0.02 x 104 M-1 s-1 (mean ± SD) The interaction between the aptamers and AT was also measured in human plasma. In a clotting assay, aptamer R6_15 accelerated clotting time by approximately 7 seconds (from 44.3 ± 0.8 to 37.3 ± 0.7 seconds). This difference in clotting time was the greatest noticed among all the other aptamer candidates. By measuring the change in AT’s fluorescence intensity, we were able to determine the aptamers’ binding capacity. The binding affinities (kd) of aptamers R6_15 and R6_19 were 65.3 ± 8.7 and 67.5 ± 14.5 nM, respectively. Truncation of R6_15 on either its 5’ or 3’ end did not increase its inhibitory activity or binding affinity towards AT. By pairing the selection data with dynamic molecular modelling, the interface of aptamer R6_15 to AT was predicted to be at the site of heparin binding, specifically at residue K114. Although these computer-generated results are not conclusive, they provide a testable hypothesis for future experimentation. Ultimately, this work provides evidence that the application of in vitro evolution has yielded a novel anti-serpin aptamer. With some modifications, the selection protocol employed in this study could be revisited to identify tighter binders and more potent inhibitors of AT. Either aptamer R6_15 or a future higher affinity AT-binding aptamer could be tested for its efficacy in reducing bleeding in vivo using mouse models of acquired hemophilia or traumatic bleeding. / Thesis / Master of Science (MSc)

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)

In vitro selection of functional RNA molecules has formed the basis for a new class of molecules termed “aptamers.” Aptamers have been selected against a wide range of molecules, ranging from simple chemical compounds to multi-cellular living organisms. The majority of selections are carried out against targets, such as proteins, that are typically composed of one type of molecule. Targets composed of multiple types of molecules (lipids, proteins, carbohydrates, etc.) are termed “complex,” and examples of successful selections against them include parasites, virions, and red blood cell ghosts. Through various properties inherent in their composition, aptamers have the potential to play a role in everything from therapeutics to broad based detection platforms. Bacterial toxins are a means by which pathogenic bacteria are able to exert an effect on a host organism. Although there are a few aptamer selections that have been carried out against toxins, there have not been any successful selections against whole bacterial cells. As some bacteria are easily grown in laboratory conditions, the possibility of their use as a biological threat agent is relatively high. Therefore, there is a need develop rapid and reliable technologies for the detection of such threats. This work details two aptamer selections carried out against both a bacterial toxin, Bacillus. anthracis protective antigen (PA), and a Bacillus subtilis vegetative cell. The selection against PA resulted in a high affinity aptamer that is capable of inhibiting the cleavage of PA. This cleavage step is the first in the pathway whereby anthrax toxin is able to exert its effect. The selection against B. subtilis vegetative cells is a proof of principle selection. B. subtilis is meant to be a surrogate for B. anthracis, which has long been regarded as a potential bio-weapon. Aptamers selected against these vegetative cells are shown to discriminate between bacterial vegetative cells of the same genus, bacteria of a different genus, and also spores produced by B. subtilis. With these selections as examples, it is hoped that the role of aptamers can continue to be expanded into viable detection systems for biological threat agents. / text

Modern cancer treatment makes extensive use of clinical imaging methods for diagnosis and response assessment. To this end, there is increasing desire to non-invasively measure various drugs and biomarkers inside a patient on a centimetre scale. Despite undeniable preclinical progress and evaluation of many techniques, few new imaging drugs are emerging into pragmatic clinical cancer imaging. There are many drug targeting strategies, including target-affinity and activation-by-target. Affinity selections can identify binders from combinatorial libraries of heteropolymers such as nucleic-acid sequences and peptides. Using this approach, in combination with next-generation DNA sequencing, I identified sequences as binders of putative cancer biomarkers. In addition, I investigated a target-activated fluorescent probe as a reporter of cancer-associated enzyme activation. Messenger RNA levels for Leucine-rich-repeat containing 15 (LRRC15) are reported to be elevated in human, breast-cancer samples. I analysed a new antibody to LRRC15, which locates this protein in genetically triggered murine breast tumours and in their lysates on Western blot. Antibody staining also showed a distinct pattern in sections of normal murine kidney, and protein expression in human breast-cancer samples. LRRC15 affinity selection of phage peptide and aptamer libraries was performed with immunopurified protein, and this identified consensus sequences. However, specific binding of the peptides or aptamers to the target was not demonstrable. Alpha folate receptor overexpression has been described in many human tumours, particularly ovarian cancer. Cell-lines to enable whole-cell selection of binders to the folate receptor were developed. Specific staining with a folate-fluorophore compound validated these. Selection of peptide and aptamer binders showed early emergence of spurious dominant sequences, triggering abandonment of this approach. The cell-lines were used to test a folate-quantum dot conjugate, with disappointing results. Matrix Metalloproteinase-9 (MMP-9) activity in cancer has previously been described and pursued as a therapeutic target. A novel probe to report activity of MMP-9 was tested using fragments of murine tissue, successfully differentiating normal murine fat pad from pieces of murine mammary tumour. Significant off-target activation was also observed, particularly with kidney. Recombinant proteins based on human MMP-2 and -13 also activated the probe. Expression and activity of equivalent enzymes in the murine tissues and tumours were assessed using RT-PCR, Western blot, immunohistochemistry and zymography, but the basis of spurious activation remains obscure. In conclusion, a new antibody identifies LRRC15 in both human and murine breast cancers, and in the murine kidney. Library affinity selections with LRRC15 and the alpha folate receptor developed consensus sequences, but were unsuccessful. An MMP-9 activated probe successfully differentiated breast tumour from normal tissue but also showed significant off-target activation. Non-invasive detection and measurement of cancer biomarkers remains an important topic, likely to see much progress in coming decades. Some of the practical difficulties in developing reagents to achieve this are discussed.

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.

Aptamers have shown a great potential due to their cheaper synthesis and easy chemical modification compared to antibodies, and have been employed in various biological assays and applications throughout the last two decades. Despite of their limitations, such as non-specific binding and low nuclease resistance, aptamers could be successfully used in the biomarker discovery and for the development of the aptamer-based imaging probes for in vitro assays. In this thesis, luminescent aptamer-conjugated nanoparticles were developed and utilized for leukemia cell detection with fluorescent microscopy. It was shown that for the bioconjugation of an aptamer with luminescent nanoparticles it is more beneficial to use carboxyl-modified nanoparticles, which results in a stable luminescence after the conjugation and the absence of unsaturated and unstable conjugates, unlike with amino-modified nanoparticles. Moreover, a cell viability assay was performed and it was revealed that aptamer-conjugated nanoparticles did not induce spontaneous apoptosis and necrosis of leukemia cells, which can be further explored with additional cytotoxicity tests, whether the aptamer-conjugated nanoparticles are biocompatible, or not. Aptamer-based biomarker discovery implies disease biomarker identification, and most commonly used methods are tedious and require a relatively high concentration of captured aptamer-target complexes. For that, AptaBiD was used in order to optimize aptamer-target identification method. Using Sgc8-aptamer, it was first shown with flow cytometry that it binds to both, healthy and malignant T lymphocytes, which requires further improvements for this aptamer to be used for leukemia detection. Among three tested detergents for the aptamer-target purification, DDM happened to be the most suitable one, due to its gentle cell lysis and solubilization properties. However, the cross-linking with formaldehyde has not positively affected the results obtained and could be replaced with photocross-linking in future experiments, which would allow to selectively cross-link an aptamer with a photomodified nucleobase with its target. Lastly, a high number of intracellular proteins identified within samples could be associated with the aptamer non-specific binding and internalization, which could be improved in future with an alternative cell fractionation with a membrane isolation approach used for the identification of transmembrane target protein.

Microtubules are a major constituent of the cytoskeleton in all eukaryotic cells. They are essential for cell morphogenesis and motility. Specifically in the dividing cells, microtubules form the spindle which segregates chromosomes. Microtubule plus ends constantly switch between phases of growth and shrinkage which is necessary for microtubule reorganization and thus their function. Importantly, microtubule dynamics are highly regulated by microtubule-associated proteins (MAPs). EB1 and Mini spindles (Msps) are unique amongst MAPs because they bind and track growing microtubule plus ends autonomously. Although essential for cell division and thus highly expressed in dividing cells, EB1 and Msps are also abundant in differentiated cells. However, to identify post-mitotic roles of proteins essential for cell division, particularly in context of a multicellular organism, is a challenge requiring new tools which I aimed to develop in my project. Since EB1 acts by recruiting MAPs to the microtubule plus ends, I generated short peptides which bind to Drosophila EB1 to block interactions with these MAPs. I showed that an EB1-MAP interaction was disturbed in Drosophila S2 cultured cells and expressing these peptides in developing Drosophila reduced fly viability. Further screening and analysis of peptides interacting with fly EB1 and its human homologues uncovered sequence determinants promoting strong binding and specificity. To uncover Msps function, I generated a msps temperature sensitive mutant and found that Msps is essential for neuromuscular function in developing Drosophila. This study showed that the regulation of microtubule dynamics has crucial functions at the whole organism level. These new tools allow the roles of microtubule regulation to be dissected in developing organisms.

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