着丝粒和端粒是真核细胞染色体的重要组成部分，他们都是由DNA和蛋白质组成的复合体。研究发现水稻的着丝粒DNA含有大量CentO卫星重复序列，而端粒DNA由富含鸟嘌呤的重复序列组成。他们的蛋白质成份在着丝粒和端粒发挥其功能的过程中起到非常重要的作用，然而对这些蛋白的了解却很少。该项研究通过使用affinity pull down技术和其他蛋白质组学方法捕捉到了一系列DNA特异性结合的蛋白；其中有86个与CentO序列结合和135个与水稻端粒重复序列结合的蛋白质被捕捉到。通过使用体外蛋白质与DNA结合验证方法，其中的一个蛋白Os02g0288200被证实了具有特异性结合着丝粒DNA的功能。同时，发现了4个水稻端粒结合蛋白。这些结果显示了affinity pull down技术能有效的应用于分离DNA特异结合蛋白，特别是着丝粒和端粒DNA结合蛋白的研究中。此外，在CenH3体外功能研究中，我们发现水稻内源CenH3蛋白对不同DNA序列的结合能力不同；与水稻rDNA序列相比，CenH3对着丝粒特异的DNA序列的结合能力更强。在研究DNA甲基化对CenH3与DNA结合能力的实验中，我们同时发现水稻CenH3蛋白对甲基化的CentO序列比未修饰的CentO序列的结合能力弱。这个结果同着丝粒功能区DNA次甲基化相吻合。 / Centromeres and telomeres are both DNA/protein complex and essential functional components of eukaryotic chromosomes. Previous researches have shown that rice centromeres and telomeres are occupied by CentO satellite repeat and G-rich telomere repeats, respectively. However, the protein components are not fully understood. DNA binding proteins associated with centromeric or telomeric DNA components will be most likely important for the understanding of centromere and telomere structure and functions. To capture DNA specific binding proteins, affinity pull down technique was applied in this research to isolate rice centromeric and telomeric DNA binding proteins. 86 proteins and 135 proteins were pulled down from CentO column and telomere repeat column respectively. One putative CentO binding protein, Os02g0288200, was demonstrated to bind to CentO specifically by in vitro assay. A conserved domain, DUF573 with unknown functions was identified in this CentO binding protein, and proven to be responsible for the specific binding to CentO sequence in vitro. Four proteins identified as telomere binding proteins in this research were studied by different groups and reported previously. These results demonstrate that the DNA affinity pull down technique is powerful in the isolation of sequence specific binding proteins and may be applicable in future studies of centromere and telomere proteins. In addition, the binding affinity of CenH3 to various forms of DNAs was analyzed by in vitro studies. The results show that rice endogenous CenH3 binds stronger to rice centromeric DNA sequences than rDNA sequence control, and prefers unmethylated CentO DNA sequence to methylated form. This phenomenon may provide explanation of the hypomethylation of centromeric DNAs in active centromeres. / Detailed summary in vernacular field only. / He, Qi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 50-55). / Abstracts also in Chinese. / List of Figures --- p.iii / List of Tables --- p.iv / List of Abbreviations --- p.v / Acknowledgements --- p.vii / Abstract --- p.viii / 摘要 --- p.ix / Chapter Chapter 1 --- LITERATURE REVIEW --- p.1 / Chapter Chapter 2 --- IDENTIFICATION OF CENTROMERE AND TELOMERE DNA BINDING PROTEINS IN RICE --- p.10 / Chapter Chapter 3 --- IN VITRO STUDIES OF CENH3 BINDING TO CENTRIMERIC DNA AND ITS METHYLATED FORM --- p.36 / Chapter Chapter 4 --- CONCLUSIONS AND PERSPECTIVES --- p.48 / References --- p.50

Graduation date: 2005

La capture d'oligonucléotides (ON) par les cellules eucaryotes, considérée comme une étape limitante de leur efficacité, implique leur endocytose. Plusieurs publications ont rapporté une capture sélective d'ON, suggérant l'intervention de récepteurs de transport. Ma thèse de doctorat a donc visé à rechercher et à caractériser d'éventuelles protéines réceptrices pour les ON qui pourraient faciliter leur endocytose. Nous avons choisi dans ce but la lignée d'hépatocarcinome différencié HepG2, où l'endocytose d'ON a été démontrée par d'autres auteurs. J'ai d'abord développé une nouvelle méthode, le « ligand blotting », et identifié une protéine transmembranaire, qui lie spécifiquement les ON, et est partiellement exposée à la surface de la cellule, trois conditions nécessaires pour un candidat-récepteur. La purification de cette protéine a donc été entreprise et une séquence partielle a pu être obtenue, mais aucune homologie n'a été trouvée dans les banques de données et il n'a pas été possible de la cloner. J'ai ensuite étudié les propriétés de l'endocytose d'ON à faible concentration, pour éviter la confusion avec l'endocytose non spécifique, appelée endocytose fluide. Dans ces conditions, l'endocytose est saturable en fonction de la concentration du traceur, atteint un plateau après environ deux heures de capture, et est sensible aux mêmes compétiteurs que la liaison d'ON à la protéine « réceptrice » identifiée en « ligand blotting ». Ces propriétés sont compatibles avec celles de l'endocytose par récepteurs. Toutefois, après intériorisation, l'ON se retrouve essentiellement à la périphérie des cellules et ne co-localise pas avec la transferrine et le dextran, marqueurs classiques des endosomes et s'accumule dans des structures denses qui ne sont pas des lysosomes. Vers la fin de mon doctorat, j'ai mis en évidence, de manière tout à fait inattendue, que la présence de la protéine « réceptrice d'ON » est liée à une infection systématique de la lignée HepG2 par M. hyorhinis ; que cette protéine n'est autre que la protéine transmembranaire invariante (p70) de cette bactérie ; et que l'infection intentionnelle par M. hyorhinis d'une autre lignée cellulaire suffit à y augmenter fortement la capture d'ON. En conclusion, je propose l'hypothèse que la capture d'ON reflète la phagocytose de M. hyorhinis ayant fixé l'ON, et discute la signification de ces observations pour la biologie de l'infection non conventionnelle par cette bactérie.

Malaria is an infectious disease caused by eukaryotic protists in the genus Plasmodium. Approximately half of the world's population is at risk of malaria. The burden of Plasmodium falciparum malaria has increased in recent years due to the emergence of resistant strains, which have even been documented in regions previously reported as malaria-free. Although malaria vaccine research has been conducted and has showed recent positive results, there still remains no effective vaccine to prevent malaria in clinical practice. According to the World Health Organization, prompt confirmation of malaria infection by microscopy and/or rapid diagnostic test (RDT) is critical to control the spreading of malaria and to prevent the evolution of drug resistant Plasmodia strains. However, malaria diagnosis remains a significant challenge as many malaria endemic regions have inadequate access to microscopy, and antibody-based RDTs are restricted by their stability under tropical temperatures and by their cost. The objective of this study was to develop a new approach to malaria diagnosis using DNA aptamers to recognise proteins encoded by Plasmodium. The research is divided into two parts. Firstly, DNA aptamers against the diagnostic markers, P. falciparum histidine-rich protein 2 (HRP2) and P. falciparum lactate dehydrogenase (PfLDH), were selected by Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Secondly, a selected PfLDH aptamer was incorporated into a gold nanoparticle detection system to develop an aptamer-nanoparticle conjugate as a new approach towards malaria diagnosis. The identified HRP2 and PfLDH aptamers were characterised by isothermal titration calorimetry (ITC) for their affinity to targets and were observed to bind with nanomolar affinity. As PfLDH aptamers were observed to have a higher affinity to their target, PfLDH, their specificities were further characterised by ITC using human lactate dehydrogenases, hLDHA1 and hLDHB. The PfLDH aptamers were shown to be highly specific to PfLDH with no observed affinity to human LDHs. After further characterisation, PfLDH aptamer 2008s was chosen for the next stage of the research to be combined with a nanoparticle as a route towards diagnostic application. In the second part of this study, PfLDH aptamer 2008s was conjugated to gold nanoparticles (AuNPs) to create aptamer-AuNP conjugates (2008s-AuNP). The aptamer-AuNP conjugates were characterised by their tolerance in different pH and salt concentration and in their sensitivity to PfLDH. This new approach of malaria diagnosis was further validated by incubating the aptamer-AuNP conjugates with various proteins and colour changes were observed specifically upon incubation with PfLDH but not with other proteins. Hence, a Plasmodium specific aptamer-AuNP conjugate to the malaria diagnostic marker, pLDH, has been developed in this research. This work lays the foundation for further development of novel rapid diagnostic tests based on nucleic acid aptamers and nanotechnology for robust and cost-effective malaria diagnosis with potential benefit not only for malaria but in a plethora of diagnostic applications. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Since there is an urgent need for development of vaccines and antiviral agents to combat influenza pandemics, this study aimed to develop influenza virus-like particles (VLPs) and aptamers targeting the virus particles as vaccine and antiviral agent candidates. Influenza VLPs containing three structural proteins of hemagglutinin (HA), neuraminidase (NA) and matrix 1 (M1) derived from influenza A/Hong Kong/01/2009 (H1N1) virus (HK/01) were constructed using a Bac-to-Bac baculovirus expression system. The expressed VLPs were purified by sucrose density gradient ultracentrifugation and characterized by Western blotting analysis and transmission electron microscopy. The immune responses and protective efficacy induced by VLPs were compared with those elicited by the clinically used Panenza vaccine in BALB/c mouse model. The results showed that two-dose vaccination with both VLP and the Panenza vaccine could confer complete protection. Single-dose vaccination with VLP could also provide 100% protection against lethal virus challenge, whereas single dose of an equal amount (based on HA content) of the Panenza vaccination just provided incomplete protection (67% survival rate) against the lethal virus challenge. Compared to the Panenza vaccination, the VLP vaccination could induce higher and broader antibody responses and higher viral specific T help (Th) cell and cytotoxic T lymphocyte (CTL) responses. Notably, a novel finding in this study is that the VLP vaccination could induce antibodies to inhibit virus release from infected MDCK cells, although the underlined mechanism needed to be further studied. These results indicated that influenza VLP might be a more effective and safe vaccine candidate which could be developed into an alternative vaccine for the control of epidemic and pandemic influenza in the future. To develop aptamers as antiviral agents against influenza, I sought to use influenza VLPs as target for ssDNA aptamer selection. After 11 rounds of selection using the systemic evolution of ligandsby exponential enrichment (SELEX),the recovered DNA molecules were PCR-amplified, gel purified and cloned into pCR-Blunt II TOPO vector for sequencing. The sequencing results showed that one aptamer Va-1 was markedly enriched, which was accounted for 59% (13/22) of the selected aptamers. Compared to the other non-enriched aptamers, the enriched aptamer Va-1 showed the highest binding affinity to the UV inactivated influenza HK/01 virus. It was also shown that the aptamer Va-1 specifically bound to the HK/01 stain while it could not bind other respiratory viruses even the PR8 strain within the H1N1 subtype. It was further demonstrated that the aptamer Va-1 could only bind to NA protein in a dose-dependent manner but not bind to HA and M1 proteins. Unfortunately, the selected aptamer did not show any antiviral effects. However, it may be potentially developed into a diagnostic and analytic agent because its binding activity was comparable with that of the commercial anti-NA antibody. In conclusion, the influenza VLPs may be a promising vaccine candidate for the control of influenza virus infection and the selected aptamer may be potentially developed into an alternative tool for influenza virus detection. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

DNA aptamers have been studied since their inception in 1990, but have only targeted membrane and serum proteins in therapeutics. Their potential as inhibitors of protein function is hampered by their inability to efficiently enter cells in order to function. Surmounting this hurdle is worthwhile since the inhibition of protein-protein interactions is not achievable by small molecule pharmaceuticals alone. Herein we target an intracellular ubiquitin ligase WWP1, which is known to complex with Schnurri3 and polyubiquitinate Runx2, thus targeting it for proteosomal destruction. Since Runx2 is the key transcriptional regulator of osteoblast differentiation, WWP1 inhibition may encourage osteoblast differentiation, and by extension force bone deposition in osteoporosis sufferers. By targeting WWP1 we attempt to intervene in intracellular protein interactions with an aptamer for the first time. To begin this effort we cloned, expressed, and purified three functionally important truncations of WWP1. The final protein pools were highly concentrated above 2 mg/mL, approximately 95% pure, and were found to be acceptably soluble after assessment in various buffers. DNA aptamers were then selected against these WWP1 truncations using the established SELEX method while monitoring the progression of the enrichment with PCR. After 12 selection rounds of increasing stringency, pools were sequenced and assessed for homogeneity and secondary structure. Several groups of enriched and identical DNA sequences were obtained with no obvious pattern in secondary structure seen between them. While focusing on sequences specific for the active site containing C-lobe, we then evaluated the aptamers for their ability to bind key functional regions of WWP1 and inhibit its function as an enzyme. For the most potent aptamer from the C-lobe pool, an Electrophoretic Mobility Shift Assay (EMSA) estimated a Ki of around 2 μM. Furthermore, a HECT ubiquitin ligase activity assay was developed to evaluate inhibition, and an IC50 of around 100 μM was found for the most inhibitory of three C-lobe aptamers. This aptamer was then transfected into SaOS-2 osteoblastic cells so that localization could be assessed with fluorescence microscopy. Surprisingly, both the C-lobe specific aptamer and a control sequence were found to enter the cells with or without the employment of transfection reagent. Moreover, approximately 60% migrated to the nucleus and remained there over a period of days, which implies diffusion through the Nuclear Pore Complex. Taken together, this work introduces an alternative approach to disease therapy by targeting intracellular proteins with aptamers, and may have significant implications for expanding the therapeutic applications of nucleic acid aptamers in the future. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

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During the past two decades, oligonucleotide analogues have drawn considerable attention as potential therapeutic and diagnostic agents. Gene silencing through "RNA interference" (siRNA) or the more mature "antisense" technology (AONs) have proven to be powerful tools for studying gene functions. Chemical modifications of these compounds are generally required to improve their "drug-like" properties such as potency, selectivity and delivery, particularly in the development of oligonucleotide-based therapeutics. Aptamers are another emerging class of oligonucleotide therapeutics and diagnostics. / This thesis focuses on oligonucleotides containing 1-(2-deoxy-2-alpha-C-hydroxymethyl-beta- D-ribofuranosyl)thymine (2'-alpha-hm-dT, abbreviated as "H") and 2'-deoxy-2'-fluoroarabinonucleotides (2'F-araN), and their applications. A major component of this work focused on the synthesis of 2'-alpha-hm-dT (H) and the first investigation of oligoribonucleotides containing this nucleoside analogue. Specifically, 2'-CH2O-phosphoramidite and 3'-O-phosphoramidite derivatives of H were synthesized and incorporated into both 2',5'-RNA and RNA chains. Incorporation of 3',5'-linked H units into a DNA, 2',5'-RNA or RNA strand led to significant destabilization of duplexes formed with unmodified RNA targets. 2',5'-Linked H units into 2',5'-RNA or RNA caused significantly less destabilization, and in fact, they were shown to stabilize the loop structure of some RNA hairpins. These results were rationalized in terms of the "compact" and "extended" conformations of nucleotides. / A series of branched RNAs (Y-shaped) related to yeast pre-mRNA splicing intermediates were synthesized incorporating both natural (i.e., ribose) and nonnatural (i.e., H, and acyclic nucleoside) branch points in order to examine the effect of sugar conformation and phosphodiester configuration on yeast debranching enzyme (yDBR) hydrolytic efficiency. The results indicate that 2'-phosphodiester scission with yDBR occurs only with a ribose-phosphate backbone at the branch point, whereas some of the H-containing branched RNAs were found to competitively inhibit yDBR hydrolytic activity. / This thesis also examines the stabilization of DNA guanine-quadruplexes (G-quadruplexes) by replacing the deoxyribose sugar by a 2-deoxy-2-fluoroarabinose. The effect of this substitution was assessed in the well-known thrombin-binding DNA aptamer d(G2T2G2TGTG2T 2G2), the telomeric DNA d(G4T4G 4) sequence and a phosphorothioate octanucleotide PS-d(T2G 4T2), all of which are known to fold into G-quadruplex structures. Stabilization of the G-quadruplexes was possible provided that the arabinose sugar was introduced at guanosine residues adopting an anti N-glycosidic bond conformation. Some of the arabinose modified thrombin-binding aptamers not only exhibited superior thermal stability and nuclease resistance, but also maintained high thrombin binding affinity. / Finally, this thesis examines the ability of DNA polymerases to recognize and utilize 2'-deoxy-2'-fluoro-beta-D-arabinonucleoside 5'-triphosphates (2'F-araNTPs) as building blocks for the synthesis of 2'-deoxy-2'-fluoro-beta- D-arabinonucleic acids (2'F-ANA). The results obtained indicate that a few DNA polymerases can synthesize 2'F-ANA and 2'F-ANA-DNA chimeras on a DNA template. Conversely, certain enzymes were shown to catalyze 2'F-ANA template-directed DNA synthesis. While it was not possible to synthesize 2'F-ANA strands on a 2'F-ANA template, it is possible for some DNA polymerases to catalyze the formation of multiple 2'F-ANA:2'F-ANA base pairs within a DNA-FANA chimeric duplex. These results suggest that it should be possible to evolve FANA-modified aptamers via SELEX.

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