The macrocyclic square planar nickel complex, [Ni II CR] 2+ , has been shown to be a useful DNA or RNA structure probe due to its highly site- and conformation specific ability to induce cleavage on exposed guanine residues via the formation of a direct guanine N7-Ni III bond. Since the postulated intermediate [Ni III CR] 3+ is unstable, the detailed mechanism is unknown. In this study, the nature of the interaction of NiCR 2+ and its oxidized products with biomolecules was investigated. A study of the conversion of [Ni II CR] 2+ between a diamagnetic square planar structure and a paramagnetic tetragonal structure in aqueous solution has shown that the conversion is affected by the identity and the concentration of the counter anion. Of the anions studied, it is clear that Br − , ClO 4 − , and CF 3 SO 3 − have a higher ability to promote the conversion to the square planar form for [NiCR] 2+ than Cl − or CF 3 COO − . The oxidation reaction of [NiCR] 2+ with either KHSO 5 or Na 2 S 2 O 8 in a molar ratio of 1/1 resulted in the same stable complex [Ni(CR-2H)] 2+ . A single crystal x-ray diffraction study gave the structure of Ni(CR-2H)(ClO 4 ) 2 . In addition, kinetic studies revealed the oxidation reaction to be first order. The six protons on the two methyl groups of the macrocyclic ligand were also found to be sufficiently labile to exhibit hydrogen/deuterium exchange. The [Ni(CR-2H)] 2+ displays a higher acidity than [NiCR] 2+ by H/D exchange. This observation supports the conjecture that there is an enhanced dπ-pπ* back-bonding effect associated with the presence of the additional imine formed in [Ni(CR-2H)] 2+ . The [NI(CR-2H)] 2+ species with KHSO 5 also displays an oxidation ability similar to [NiCR] 2+ with KHSO 5 in the reaction with a 17 base pair synthetic oligonucleotide. This implies that [Ni(CR-2H)] 2+ is not just an oxidation product of [NiCR] 2+ , but may also play an important role in the reaction with guanine residues in oligonucleotides. The reactions of [NiCR] 2+ or [Ni(CR-2H)] 2+ with linoleic acid under a high concentration of Ni complexes (3.21 × 10 −3 M, 200 fold over linoleic acid) resulted in the unexpected reduced nickel complexes, (Ni 0 (CR-4H)-H + ) − - m/z 311.1 and (Ni 0 (CR-2H)-H + ) − - m/z 313.1, instead of the hydroperoxide product (HpODE-H + ) − - m/z 311.2.

<p> Cationic polyelectrolytes have attracted growing attention in the field of non-viral oligonucleotides (ONs) deliveries because of their ability to bind ONs by electrostatic interactions for efficient cellular uptake. However the formation of electrostatic polymer/ONs complexes and their biological effects are still poorly understood. The relationships between polymer structure and complexation performance have not been well established. The objectives of this research are to synthesize and characterize well-defined and well-controlled cationic polyelectrolytes and to evaluate the effects of polyelectrolyte chain properties on ONs complexation. Poly(2-(dimethylamino) ethyl methacrylate) (polyDMAEMA) and its derivatives are used as the polymer candidate. A fluorescein-labeled oligonucleotide, 5 '-FGCGGAGCGTGGCAGG-3' (F: fluorescein), is used as the oligonucleotide candidate.</p> <p> Low-molecular-weight cationic polyDMAEMA samples having narrow molecular weight distribution were synthesized by living anionic polymerization (LAP) and atom transfer radical polymerization (ATRP) methods. Fully charged polyDMAEMA quats were prepared by sequential quaternization of polyDMAEMA samples, as well as by direct ATRP of the quaternized DMAEMA monomer. An aqueous GPC calibration method was first developed for the characterization of these cationic polyelectrolytes. It was found that the type of counter-ion has little effect on the hydrodynamic volume of polyDMAEMA quat. Therefore the dimethyl sulfate salt of polyDMAEMA provided a reliable calibration standard for other types of quaternized DMAEMA homopolymers.</p> <p> Cationic block copolymers of polyDMAEMA with 2-hydroxyethyl methacrylate (HEMA) and polyethylene glycol (PEG) were also prepared by ATRP. It was found that the order of monomer addition, solvent type, temperature, and molecular weight of macroinitiator have significant effects on the living feature of the polymerization. Well-controlled block copolymers were obtained when polyHEMA was used as the macro initiator.</p> <p> The complexation capability of the prepared polyelectrolytes with oligonucleotides (15 mer) was evaluated by a fluorescence technique. It was found that the complexation performance depends on polymer molecular weight, charge density, and counter-ion type, as well as polymer concentration and block composition. The polymer sample that has double molecular weight of the ONs gave the optimal complexation performance.</p> / Thesis / Master of Applied Science (MASc)

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No description available.

Development of chiral ligands capable of achieving high selectivity for various asymmetric catalytic reactions has been an important aim of both academia and industry. Nature is capable to selectively catalyze chemical reactions by using enzymes. An ideal catalyst would combine the selectivity of nature and the reactivity of man-made catalysts based on transition metal complexes. The two biomolecules chosen to achieve this are DNA and PNA. DNA is a chiral molecule with high binding selectivity towards small molecules and has been used as ligand for asymmetric catalysis. PNA is an achiral structural analogue of DNA that can form duplexes with DNA. To produce DNA based catalysts it is necessary to introduce a ligand such as a phosphine that will strongly coordinate to transition metals. To achieve this, functionalized linkers need to be introduced into a DNA strand, to covalently couple the phosphine moiety at a specific location of the DNA strand. Amine linkers and several modified nucleosides have been prepared containing thiol and amine functionalities and some of them were successfully introduced into DNA strands to function as linkers for the introduction of phosphine functionalities. Those strands were purified and an adequate procedure was developed for their analysis by MALDI-TOF. Diphenylphosphino carboxylic acids have been coupled to amine modified deoxyuridines by amide bond formation. The same coupling method has been used for oligonucleotides. DNA strands containing phosphine moieties were characterized by MALDI-TOF and ³¹P NMR spectrometry. ³¹P NMR spectroscopy was also used to confirm coordination of a phosphine modified 15-mer to [PdCl(η³-allyl)]₂. The phosphine modified nucleobases were also tested as ligands for palladium catalyzed allylic alkylation and allylic amination with diphenylallyl acetate as substrate although no enantioselectivity was observed. A PNA monomer was also modified with a bidentate sulfur protected phosphine and successfully introduced into a short PNA strand using manual solid phase synthesis. This strand was analyzed by MALDI-TOF. Moreover, preliminary studies were performed to test the use of aptamers as scaffolds for targets containing a ligand functionality.

Ngan, Kit Shan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 176-187). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

La myopathie myotubulaire est une maladie musculaire congénitale très sévère. Le laboratoire d’accueil a démontré que les échantillons de muscle de patients atteints de cette maladie ainsi que le modèle murin présentent une surexpression de DNM2, alors que sa réduction par croisement génétique améliore les signes cliniques et histologiques de la maladie. Le but de ce travail consistait à développer, tester et valider des composés injectables qui ciblent DNM2 et diminuent son niveau. Deux approches thérapeutiques ont été développées l’une basée sur l’utilisation de virus adéno-associés (AAV) exprimant des shRNA, l’autre sur les oligonucleotides antisens (ASO). L’injection des vecteurs AAV-shDnm2 ou bien les ASO-Dnm2 pouvait corriger les défauts histologiques et fonctionnels des muscles des souris myopathes.Les résultats obtenus montrent le potentiel thérapeutique de la réduction de DNM2, et présente une nouvelle approche pour le traitement de la myopathie myotubulaire. / Myotubular myopathy is a severe muscle disease. We previously have shown that muscle specimens of both patients and the mouse model presented an overexpression of DNM2, while its genetic reduction prevents the development of the muscle phenotypes. The aim of this work was to develop, test and validate deliverable compounds. Two therapeutic approaches were used. Injection of antisense oligonucleotide or adeno-associated virus expressing shRNA restores a normal lifespan with improved muscle structure and function of the myopathic mice. These results demonstrate that therapeutic potential of reduction of DNM2 level and provides an attractive therapeutic strategy that could be applied to treat myotubular myopathy.

An unprecedented use of high-throughput sequencing for routine monitoring of SARS-CoV-2 viruses in patient samples has created a dataset of over 6 million SARS-CoV-2 genomes. To monitor genomes, deposited in the GISAID database, and to track the continuous sequence evolution of molecular assay oligonucleotide target sequences. A simple pipeline tool for non-experts was developed to mine this database for nucleotide changes in oligonucleotides and tested with the long oligonucleotides of a Recombinase polymerase amplification (RPA) assay targeting the RNA-dependent RNA polymerase (RdRP) gene of the SARS-CoV-2. Results indicate the emergence of a single nucleotide change in the reverse oligonucleotide from 0.03 to 26.23% (January to May 2021) in Alpha variant genomes, which however reduced to 17.64% by September after which the Alpha variant was completely displaced by the Delta variant. For all other variants, no relevant nucleotide changes were observed. The oligonucleotide screening pipeline allows efficient screening of nucleotide changes in oligonucleotides of all sizes in minutes.

A photolabile terpolymer containing 6-nitroveratyloxycarbonyl (NVOC) protected amine, epoxy and trimethoxysilyl functionality in 1:3:2 monomer ratio was synthesized to template glass surfaces for specific site directed coupling of non-probe oligomers and probe oligonucleotides. Non-probe oligomers were introduced to the surface to control the environment of the probes by reducing probe-to-probe and probe-to-surface interactions. The trimethoxysilyl group served as the anchoring site for the terpolymer to be covalently bound to glass and silicon wafers. Amine terminated non-probe oligomers were coupled to the epoxy sites and thiolated 19-mer SMN1 probes were directed to the deprotected amine sites via the heterobifunctional linker, sulfosuccinimidyl-4-[maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC). Characterization of the terpolymer was done using 1H NMR, 13C NMR, MALDI-ToF and elemental analysis. NVOC deprotection was monitored by UV absorption, and surface characterization of the bound terpolymer on silicon wafers was investigated with XPS, ToF-SIMS, ellipsometry and static contact angle. Neutral polyethylene glycol (PEG), negatively charged methacrylic acid (MAA) oligomer and dC20 oligonucleotides were used as non-probe oligomers. The probe density on the surface was estimated to be 2.2 ± 0.3 x 10^12 molecules/cm2 and the presence of the oligomers on the surface did not significantly affect probe immobilization efficiency. The mixed films were functional for target hybridization and its selectivity towards partially-mismatched targets was investigated at different solution pH, ionic strength and temperature. It was demonstrated that pH can be tuned to ameliorate non-specific adsorption and ionic strength governed the selectivity of the surfaces. Improved selectivity was achieved at high salt concentration (1 M NaCl) on PEG and dC20 mixed films at room temperature. The MAA surface did not show significant improvements in selectivity. This indicated that charge of the oligomers does not dominate control of selectivity. The results suggested that the terpolymer construct played a role in depression of the melting temperature of the hybridized duplex to within 5 to 10 oC of room temperature. With the melting temperature shifted closer to room temperature, it is possible to improve selectivity for room temperature detections of single nucleotide polymorphism.

The Hepatitis B core antigen (HBcAg) has been proposed to be an ideal candidate for use as an adjuvant due to its immunogenicity, and tolerance to manipulations such as insertions of epitopes or covalent attachment of ligands. HBcAg is a complex macromolecule containing protein and nucleic acid. We investigated the effect of the removal and reconstitution of nucleic acids upon its structure. It’s been shown that the RNA content of hepadnavirus core antigens can be reduced significantly, but not be completely removed. Following removal of some of the RNA, antigens retain the ability to bind added nucleic acids, in particular, "immune-enhancing" synthetic oligonucleotides without affecting the structure of antigen or disrupting its ability to spontaneously self-assemble into core particles. The removal and addition of nucleic acids was successfully applied to an altered woodchuck core antigen, with a nucleic acid-based malaria epitope addition, giving rise to a potential vaccine adjuvant platform for malaria.