Mercury exposure has been related to neurological diseases and poisoning. Quantification of mercury in biological fluids, such as serum or urine is an important diagnostic method for mercury exposure. We have developed an aptamer-encapsulated porous phospholipid nanoshell (PPN) sensor for sensing mercury in urine using a modified 15-mer single strand DNA.1 The probe is protected from DNAse and other biofouling species by encapsulation within the porous liposomes composed of mixed phospholipids, allowing direct application of the aptamer in biological fluids containing DNAse and other biofouling materials. The encapsulated sensor was directly tested in urine samples at physiological pH. We were able to detect below 100 ppb (500 nM) Hg2+ in urine (urine mercury threshold set by Biologischer Arbeitstoff Toleranz Wert or BAT)1 with no sample preparation other than pH adjustment. These results suggest that porous phospholipid nanoshells (PPNs) can serve as a general-purpose protection scaffold for biological sensing.

Multivalent aptamer refers to a technique that joins two or more identical or different types of aptamer monomers together, with or without the presence of structural or other functional elements. As a rapid and easy method for fabricating the multivalent aptamer constructs, rolling circle amplification (RCA) has attracted great attention in recent decades. The incorporation of properly designed structural elements, such as intra-molecular spacers, have been shown to greatly enhance the efficiency of the multivalent aptamer system [1]. The objective of this current study is to systemically investigate the effect of different lengths of poly thymine spacer designs (polyT, from no spacer/NT, 5T, 10T, and 15T) on the capturing performance of RCA-generated multivalent system. To achieve this, we designed four circular probe templates by inserting zero, five, ten, and fifteen adenine bases (polyA). These polyA domains in the circular probe template are complementary to polyT with respective lengths in between the adjacent aptamers on the resultant RCA products (RCAPs). We found that the resultant RCAPs with length shorter than 10T showed a lack or low ability to capture target cells E.Coli O157:H7. When spacer lengths reach or exceed 10T, the capturing performance of the respective multivalent aptamer chain is significantly enhanced. This phenomenon can be explained by larger hydrodynamic sizes and less nonspecific secondary structures observed in RCAP with spacer length no less than 10T. Moreover, we found that there is also a trade-off that the number of polyA bases added into the circular probe template can significantly impair the efficiency of RCA reaction in respective to cyclization yield and amplification rate. The results of this research explain with details how the design of spacer affects RCA reaction efficiency and RCAPs’ capturing performance, which provides ideas in designing an efficient RCA-generated multivalent system.

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 Coronavirus disease 2019 (COVID-19) pandemic has highlighted the critical need for accurate and sensitive diagnostic tools for detecting the SARS-CoV-2 virus. The nucleocapsid (N) protein is essential for virus replication and plays vital roles in virus assembly, packaging, and RNA transcription. This protein is a crucial component of the viral particle and is less prone to mutations than the other essential proteins in SARS-COV-2. All of these make the N protein a reliable target for virus detection. Aptamers, single-stranded oligonucleotides that can specifically bind to target molecules, have been proposed as a promising alternative to antibodies for detecting and treating viral infections. This study aimed to select DNA aptamers against the N protein of SARS-CoV-2 using capillary electrophoresis (CE) and validate the binding specificity of the aptamers. After selecting seven clones, a preliminary binding validation was performed, and the two best binding clones were identified as ECK4 and ECK6. The structures of the aptamers were then modified by removing the primer regions from the original sequence, and the binding capacity of the truncated aptamers was confirmed. Dissociation constant (KD) values were calculated to provide further supportive information for the quality of the two clones. Additionally, Biolayer interferometry (BLI) was used to calculate Apparent KD as an alternative technique and provided consistent results with CE. Our results demonstrate the successful selection of aptamers for the N protein of SARS-CoV-2 using CE-SELEX. Confirming the aptamers' binding capacity to N protein paves the way for developing aptamer-based diagnostics for COVID-19.

The highly specific recognition processes between biomolecules mediate various crucial biological processes. Uncovering the molecular basis of these interactions is of great fundamental and applied importance. This research work focuses on understanding the interactions of several biomolecular recognition systems and processes that can provide fundamental information to aid in the rational design of sensing and molecular recognition tools. Initially, a reliable and versatile platform was developed to investigate biomolecular interactions at a molecular level. This involved several techniques, including biomolecule functionalization to enable attachment to self-assembled monolayers as well as atomic force microscopy (AFM) based force spectroscopy to uncover the binding or rupture forces between the receptor and ligand pairs. It was shown that this platform allowed determination of molecular binding between single molecules with a high specificity. The platform was further adapted to a general sensing formulation utilizing a group of flexible and adaptive nucleic acid recognition elements (RNA and DNA aptamers) to detect specific target proteins. Investigation of interactions at the molecular level allowed characterization of the dynamics, specificity and the conformational properties of these functional nucleic acids in a manner inaccessible via traditional interaction studies. These interactions were then adapted to aptamer-based detecting methods that at the ensemble or bulk scale, specifically taking advantage of mechanisms uncovered in the biophysical study of this system. A quartz crystal microbalance (QCM) was used to detect protein targets at the bulk level and the affinities and binding kinetics of these systems were analyzed. Along with AFM-based force spectroscopy, ensemble-averaging properties and molecular properties of these interactions could be correlated to contribute to bridging the gap across length scales. Finally, more broadly applicable sensing platform was developed to take advantage of the unique properties of aptamers. DNA was employed both as a carrier and as a molecular recognition agent. DNA was used as a template for nanoconstruction and fabricating unique shapes that could enhance the aptamer-based molecular recognition strategies. With aptamers tagged to distinct nanoconstructed DNA, a novel shape-based detecting method was enabled at the molecular level. The results demonstrated that this is a flexible strategy, which can be further developed as ultrasensitive single molecule sensing strategy in complex environments.

PhD, Virology, Faculty of Health Sciences, University of the Witwatersrand / HIV-associated cardiomyopathy is a multifactorial disease with a broad spectrum of aetiologies that arise due to chronic immunosupression during HIV infection. The intricate relationship between HIV infection and cardiac co-morbidity was investigated with the aid of HIV-neutralizing aptamers. These synthetic nucleic acid ligands with antibody-like properties are molecular tools with multifunctional applications ranging from drug discovery to diagnostics and therapeutics. The advent of the HIV/AIDS pandemic has naturally married the field of HIV therapy and diagnostics with that of aptamer technology. By employing a HIV-1 neutralizing aptamer, named UCLA1, raised against the viral surface envelope glycoprotein 120, I dissected some of the pathways leading to cardiomyocyte apoptosis in a cell culture system. In chapter one I investigated the potential cytotoxic effects of UCLA1 by comparing it against a panel of 17 antiretrovirals (ARVs) in clinical use with the goal of establishing a safety portfolio geared towards its use as a therapeutic agent. Using cultured human cardiomyocytes and primary peripheral blood mononuclear cells (PBMCs), I selected some of the major biological markers of ARV-induced cytotoxicity and found no measurable deleterious effect, especially when compared to other ARVs used in the same study. In chapter two, the permissiveness of cardiomyocytes to HIV infection as well as the relationship between virus-host interaction and caspase-mediated apoptosis were investigated. Non-productive, receptor and tropism-independent infection was observed, which was arrested after the reverse transcription stage. However, interaction between the virus gp120 and the host’s CXCR-4 chemokine receptor preferentially activated caspase-9 triggering robust mitochondria-mediated apoptosis. A shift from mitochondrial-initiated, caspase-9 mediated to Fas-ligand initiated, caspase-8 mediated was observed when CM were co-cultured with HIV-infected MDM. UCLA1 protected against caspase-9 mediated vii apoptosis but not caspase-8 mediated. Finally in chapter three I provided answers for the shift in caspase activation by showing that supraphysiological levels of IL-1β and IL-6 during HIV infection of MDM augment the effects of tumor necrosis factor (TNF). These observation provide new insight into the complex pathophysiology of HIVCM and highlight the potential of UCLA1 as a novel therapeutic agent to fight HIV and some of its associated diseases.

abstract: The highly predictable structural and thermodynamic behavior of deoxynucleic acid (DNA) and ribonucleic acid (RNA) have made them versatile tools for creating artificial nanostructures over broad range. Moreover, DNA and RNA are able to interact with biological ligand as either synthetic aptamers or natural components, conferring direct biological functions to the nucleic acid devices. The applications of nucleic acids greatly relies on the bio-reactivity and specificity when applied to highly complexed biological systems. This dissertation aims to 1) develop new strategy to identify high affinity nucleic acid aptamers against biological ligand; and 2) explore highly orthogonal RNA riboregulators in vivo for constructing multi-input gene circuits with NOT logic. With the aid of a DNA nanoscaffold, pairs of hetero-bivalent aptamers for human alpha thrombin were identified with ultra-high binding affinity in femtomolar range with displaying potent biological modulations for the enzyme activity. The newly identified bivalent aptamers enriched the aptamer tool box for future therapeutic applications in hemostasis, and also the strategy can be potentially developed for other target molecules. Secondly, by employing a three-way junction structure in the riboregulator structure through de-novo design, we identified a family of high-performance RNA-sensing translational repressors that down-regulates gene translation in response to cognate RNAs with remarkable dynamic range and orthogonality. Harnessing the 3WJ repressors as modular parts, we integrate them into biological circuits that execute universal NAND and NOR logic with up to four independent RNA inputs in Escherichia coli. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2019

The thesis aimed to develop and characterize a novel detection approach, which we termed aptaPCR exploiting nucleic acid aptamers as combined recognition and reporter biocomponents for the ultrasensitive detection of analytes. 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. As compared to other natural and synthetic receptor, aptamers possess unique chemical and biochemical characteristics, such as: a well known chemistry, remarkable stability, an ability to be selected against virtually any target even in non-physiological conditions

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

<p>Stroke is the leading cause of morbidity and the third leading cause of death in the United States. Over 80% of strokes are ischemic in nature, produced by a thrombus occluding the cerebral circulation. Currently, there is only one pharmacologic treatment FDA approved for ischemic stroke; recombinant tissue-type plasminogen activator (rtPA). Unfortunately, thrombolysis with rtPA is underutilized, as it must be administered within three hours of symptom onset and it is not uncommon for treatment to result in intracranial hemorrhage. For these reasons, safe and effective treatments of stroke are a medical necessity. </p><p>Aptamers are an attractive emerging class of therapeutic agents that offer additional safety because their activity can be reversed with administration of a complimentary oligonucleotide. Accordingly, I hypothesized that aptamers could be used to treat acute ischemic stroke. First, an antithrombotic aptamer previously generated against coagulation factor IXa was used in a murine model of middle cerebral artery occlusion. Upon factor IXa aptamer administration following stroke, neurological function and inflammatory profiles were improved. Moreover, mice previously treated with the aptamer, followed by induction of subarachnoid hemorrhage, had severe mortality levels and hemorrhage grades that were mitigated by administration of the aptamer's matched antidote.</p><p>Second, I generated aptamers against the antifibrinolytic protein plasminogen activator inhibitor-1 (PAI-1), under the hypothesis that aptamer inhibition of PAI-1 would result in a reversible thrombolytic agent. However, after further testing, the aptamers were not found to disrupt the interaction between PAI-1 and its target proteases. Instead, the aptamers were shown to prevent PAI-1 binding to vitronectin, which translated to restoration of breast cancer cell adhesion in an environment of PAI-1 mediated detachment. </p><p>Therefore, aptamer inhibition of factor IXa has demonstrated efficacy in improving outcome following stroke, and should life-threatening hemorrhage arise, an antidote specific to the interventional agent is able to decrease not only hemorrhage grade, but also mortality. This may result in a safer stroke therapy, while a novel aptamer generated against PAI-1 may have application as an antimetastatic agent, which could be used as adjuvant therapy to traditional breast cancer treatment.</p> / Dissertation