Chemistry

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This is the collection for the University of Waterloo's Department of Chemistry.

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Browsing Chemistry by Author "Dieckmann, Thorsten"

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    Biochemical Characterization and Optimization of RNA Aptamers Bound to Hoechst Dye Derivatives
    (University of Waterloo, 2023-08-09) Evans, Natasha; Dieckmann, Thorsten
    Ribonucleic acid (RNA) aptamers are single-stranded nucleic acids that are typically selected through a process known as Systematic Evolution of Ligands by EXponential Enrichment (SELEX). These oligonucleotides can bind to small target molecules, including proteins and chemical toxins, with high specificity and affinity. They can be further modified with recognition elements, such as light-up fluorophore molecules, to become aptamer-based biosensors. In the year 2008, Sando et al. selected for an RNA aptamer that is capable of specific binding to a Hoechst dye derivative with bulky tert-Butyl (tBu) groups, which prevents the non-specific binding to DNA sequences. The group performed some preliminary biochemical characterization experiments; however little information is currently known about the specific molecular interactions between the optimized Aptamer II-mini3-4 sequence and the tBu Hoechst dye. Therefore, this research project aimed to develop a complete biochemical profile of the SELEX-selected 71-nucleotide (nt) Aptamer II sequence and the optimized 29-nt Aptamer II-mini3-4 sequence. In addition, new RNA aptamer variants were developed within this research project to not only gain a deeper understanding of the aptamer’s folded conformation and binding interactions with the tBu Hoechst dye, but to also optimize the aptamer system further. In Chapter 2 fluorescence emission experiments were performed to determine if binding was present between the RNA aptamer variants and the derivative tBu Hoechst dyes. The fluorescence enhancement of each bound complex was also compared and evaluated to understand binding interactions within the aptamer systems. Moreover, fluorescence titration assays were conducted to estimate the binding affinities for between the interacting molecules. In Chapter 3 native polyacrylamide gel (PAGE) experiments were conducted to determine the conformation of the tBu Hoechst-bound and unbound conformations of the RNA aptamer variants. In addition, isothermal titration calorimetry (ITC) experiments were employed as an alternative method to determine the dissociation constants of the studied aptamer systems. It was also used to obtain the thermodynamic and kinetic properties of each of the tBu Hoechst-binding RNA aptamers. The information gained within this research project was intended to lay the groundwork for future work including the structure elucidation of the Aptamer II-mini3-4 RNA sequence bound with the tBu Hoechst dye through nuclear magnetic resonance (NMR) spectroscopy techniques. It was also intended to aid in optimizing the aptamer system for its development as a building block for an aptamer-based biosensor that can be used for potential commercial applications.
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    Biochemical Characterization of Fluorophore-Binding Nucleic Acids
    (University of Waterloo, 2022-09-27) Tormann, Alexandra; Dieckmann, Thorsten
    Aptamers, single stranded RNA or DNA that bind to target ligands with high affinity and specificity, have a wide range of applications, from therapeutics to biosensing, which makes biochemical characterization of the utmost importance. This research aimed to implement various analytical instrumentation methods for the biochemical characterization of DNA Mango aptamers sequences 2a, 4a, and 6b, to TO1-3PEG. In Chapter 3, fluorescence spectroscopy titrations were conducted. An equilibrium dissociation constant of 160.6 nM was calculated for the 4a aptamer sequence in sodium phosphate buffer. The increase in fluorescence enhancement was weaker than anticipated; therefore, further investigation is required. Isothermal titration calorimetry experiments were also conducted, summarized in Chapter 4. The binding affinity for the DNA Mango aptamer sequences was determined. It was concluded that no binding interactions between the 2a or 6b aptamer sequences and the target ligand were observed. The 4a sequence was found to bind weakly to the target ligand in both a sodium phosphate and a HEPES buffer. Native PAGE studies were also completed, outlined in Chapter 5. The native PAGE experiments indicated the presence of multimeric structures with structural heterogeneity in both the 4a and 6b sequences. A monomeric structure was also observed in the 4a aptamer. Chapter 6 investigated the binding affinity of two DNA Mango aptamer sequences, 4a and 6b, to the target ligand via Surface Plasmon Resonance. Numerous experimental conditions were explored, but binding interactions were not detected for the explored aptamers, therefore either eluding to a weak binding interaction, or the absence of binding altogether.
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    Determining the Structures and Properties of Biologically-relevant Ions
    (University of Waterloo, 2018-08-09) Psutka, Jarrod; Hopkins, W. Scott; Dieckmann, Thorsten
    Gas phase studies of biologically relevant ions are increasing in popularity due to the possibility of high throughput analysis requiring minimum sample concentrations. This thesis explores the potential of differential mobility spectrometry-mass spectrometry (DMS-MS) in combination with quantum chemical calculation methods to probe the structures, energetics, and dynamics of three distinct classes of biomolecules. The first project outlines the use of DMS-MS to separate and identify protonated forms of methylated and unmethylated nucleobases to gain a fundamental understanding of their gas phase properties in relation to their role in nucleic acids. Next, DMS-MS and calculations were conducted for a large RNA system, the Varkud Satellite ribozyme active site loop VI, to study differences between its active and inactive conformations, especially through the use of negative mode hydrogen-deuterium exchange. Finally, DMS-MS was used to identify transformation products of trimethoprim, an antibiotic often found in environmental wastewaters in a reliable and efficient method. Ultimately, DMS-MS and quantum calculations have been shown to be a powerful analytical tool to investigate structures and properties of biomolecules. The methodologies described herein can have an impact in a wide variety of industries, from drug discovery to environmental wastewater cleanup.
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    Investigation of the Presence of a G-quadruplex In the Hoechst Dye Binding DNA Aptamer
    (University of Waterloo, 2022-04-22) Egerdeen, Kyla; Dieckmann, Thorsten
    Aptamers are nucleic acid sequences that have been selected to bind to a ligand with high affinity and selectivity. Aptamers have great potential for use in various biotechnological and medical applications but understanding their structure is important to fully maximize their potential. In 2007 Sando et al. selected for an aptamer that targeted a tert-butyl (tBu) Hoechst dye developed in their lab. No formal studies were performed on the aptamer, but the team generated a predicted secondary structure that included a loop-stem element. When this project began, it was noted that the Hoechst DNA aptamer sequence was rich in guanines, creating the potential for the formation of a G-quadruplex structure. The research presented in this thesis aims to provide structural information regarding the Hoechst DNA aptamer. In Chapter 2, various biomolecular methods were used to better characterize the Hoechst DNA aptamer and variations of the Hoechst DNA aptamer. First, computational methods were used to model potential G-quadruplexes. Then, fluorescence was used to prove that the aptamer was binding to its ligand and to determine dissociation constants (Kd). Next, native gels provided evidence that the Hoechst DNA aptamer was forming a multi-stranded structure. Finally, circular dichroism (CD) suggested that the Hoechst DNA aptamer formed a G-quadruplex structure. In Chapter 3 nuclear magnetic resonance (NMR) was employed to gain structural information regarding the DNA aptamer bound to the tBu Hoechst dye. 1D Proton NMR experiments further proved that the DNA aptamer was binding to the tBu Hoechst dye, and further supported the potential presence of a G-quadruplex. Additionally, it was possible to tentatively assign the tBu peaks within the NMR spectra. Future work could provide additional structural information and could eventually lead to the elucidation of a solved structure for the DNA aptamer bound to the tBu Hoechst dye.
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    NMR Studies of Protein and Peptide Structure and Dynamics
    (University of Waterloo, 2016-04-18) Piazza, Michael; Dieckmann, Thorsten
    Calmodulin (CaM) is a small, acidic cytosolic calcium binding protein that responds to increases in intracellular Ca2+ concentrations. It is proposed to be involved in binding to and regulating over 300 functionally and structurally diverse proteins. It is comprised of an N- and C- terminal lobe separated by a highly flexible central linker region. Each of these lobes contains two EF hand motifs that are each capable of binding to one Ca2+-ion. CaM is found to exist primarily in two states: the Ca2+-replete form, holoCaM, or the Ca2+-deplete form, apoCaM. Both forms of CaM are able to bind to target proteins. CaM also undergoes post translational modifications that play a role in its regulation of target proteins. An important target of CaM are the nitric oxide synthase (NOS) enzymes. NOS catalyzes the conversion of L-arginine to L-citrulline and nitric oxide (·NO). Three isoforms of NOS are found in mammalian cells: endothelial (eNOS); neuronal (nNOS); and inducible (iNOS). All three isoforms of NOS are homodimeric and comprised of an N-terminal heme domain, containing the active site, and a C-terminal flavin-binding domain containing FAD-, FMN-, and NADPH- binding sites, linked together by a CaM-binding region. The nNOS and eNOS isoforms are constitutively expressed and are Ca2+-CaM-dependent. In contrast, iNOS is regulated at the transcriptional level and is Ca2+-independent. NOS is also found to be regulated through the phosphorylation and de-phosphorylation of key residues, specifically Thr 495, which is found in the CaM-binding domain. The exact mechanism of how CaM activates NOS is not fully understood. Studies have shown CaM to act like a switch that causes a conformational change in NOS to allow for the electron transfer between the reductase and oxygenase domains through a process that is thought to be highly dynamic. This thesis is focused on the structure and dynamics of CaM and CaM mutant constructs bound to the target peptides of the NOS CaM-binding domain at saturating and physiological concentrations of Ca2+. To investigate the structural and functional effects that the phosphorylation of Thr495 of eNOS may have on eNOS activation by CaM, the solution structure of CaM bound to the peptide comprising the eNOS CaM-binding domain phosphorylated at Thr495 was determined. To investigate the Ca2+-dependency of CaM binding NOS, nuclear magnetic resonance (NMR) studies were performed at various free Ca2+ concentrations to determine the structure and dynamics of NOS and CaM interactions at physiological Ca2+ concentrations. The results illustrate that structures of CaM-NOS complexes determined at saturating Ca2+ concentrations cannot provide a complete picture because the differences in intramolecular dynamics become visible only at physiological Ca2+ levels. Numerous studies use CaM mutants incapable of binding Ca2+ in either the N- or C-lobe to mimic apoCaM, with some of these studies reporting functional differences when comparing the mutant and apo forms of CaM. We investigated the structural consequences of these mutations by determining the residue-specific chemical shift perturbations induced by these mutations. This was accomplished by determining the full backbone chemical shift assignments of three Ca2+-deficient CaM mutants in the absence and presence of Ca2+, and investigating their interaction with the iNOS enzyme through determination of the solution structure of a Ca2+-deficient CaM mutant with iNOS. The use of NMR spectroscopy allowed for the determination of high resolution structures of these complexes. 15N relaxation and H/D exchange experiments also allowed for the analysis of the structural dynamics occurring in these complexes. NMR spectroscopy is an efficient method for studying the dynamics and structures of protein-protein and protein-peptide complexes.
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    Selection and characterization of an aptamer to a thiazole orange derivative for use in a fluorescence-based biosensor
    (University of Waterloo, 2023-08-18) La, Volition; Dieckmann, Thorsten
    Aptamers are short single-stranded DNA or RNA sequences that have been selected in vitro to have a high binding affinity to a specific target molecule. One class of aptamers can bind to small non-fluorescent molecules to result in a dramatic fluorescence enhancement, once bound. These aptamer sequences, along with their cognate fluorogenic molecules, are used as light-up probes. RNA light-up probes are used to monitor sub-cellular localization of RNA in cell with the RNA aptamer Mango I being notable for having a high affinity along with excellent fluorescence enhancement. The usage of RNA aptamers in biosensing application is limited due to the fact that the degradation of RNA due to the pervasive RNase is a key factor in the sensors shelf life. For this reason, DNA aptamers are the choice for usage in biosensing applications. In chapter 2, a new DNA aptamer, named 02a, that binds to the target TO1-biotin was isolated after nine rounds of Systematic Evolution of Ligands by EXponential enrichment (SELEX). The selection was performed on streptavidin coated beads with the target bound to the surface with free dye in solution in higher selection rounds to select for slower off-rate binding. Using Next Generation Sequencing (NGS), the library after the 4th and 9th rounds of selection were sequenced to identify enriched sequences. The 02a sequence family emerged showing superior fluorescence enhancement, 136.5 times more than unbound TO1-biotin, as well as high affinity, with a KD of 98.7 nM, for the target, when compared to other families obtained by NGS analysis. In chapter 3, several sequence families were studied to discern structural information to better understand the binding interaction. Using several primary and secondary sequence analysis tools, a three-tiered G-quadruplex motif for the families was predicted. For the 02a aptamer, it was found that binding and fluorescence enhancement was attenuated in the absence of K+ and that the region outside the predicted quadruplex gives access to potassium-dependent fluorescence enhancement conformers. In chapter 4, biosensing approaches were evaluated for their use in the detection of the biomarker IgE. An aptamer for IgE, D17.4, was used in several sensing platforms such as Localized Surface Plasmon Resonance (LSPR), Electrophoretic Mobility Shift Assay (EMSA), Förster Resonance Energy Transfer (FRET), and Lateral Flow ImmunoAssay (LFIA) as well as in a modular aptamer configuration and was used to evaluate the detection of isolated IgE and IgE in human serum. Although the detection of IgE was possible in most set-ups, complex optimization steps are needed for optimization for commercial use, especially when using human serum samples. Chapter 5 summarizes all of the findings and future work is discussed. Overall, in vitro selection was used to select for a DNA aptamer for the thiazole orange derivative TO1-biotin which was then characterized to have a quadruplex motif. This novel aptamer, 02a, is a useful fluorescent module for biosensing applications and has superior characteristics than other published light-up DNA aptamers