Nucleic acid probes against intracellular targets can find essential applications in biodosimetry. This thesis describes the development of multiple nucleic acid probes, including aptamers and molecular beacons, against radiation-responsive intracellular molecular targets via microfluidic technology. Aptamers are single-stranded oligonucleotide molecules that bind with high affinity and specificity to a wide range of target molecules. The method of systematic evolution of ligands by exponential enrichment (SELEX) plays an essential role in the isolation of aptamers from a randomized oligonucleotide library. To date, significant modifications and improvements of the SELEX process have been achieved, engendering various forms of implementation from conventional SELEX to microfluidics-based full-chip SELEX. While full-chip SELEX is generally considered advantageous over conventional SELEX, there has not yet been a conclusive comparison between the methods. Herein, we present a comparative study of three SELEX strategies for aptamer isolation, including those using conventional agarose bead-based partitioning, microfluidic affinity selection, and fully integrated microfluidic affinity selection and PCR amplification. Using immunoglobulin E (IgE) as a model target molecule, we compare these strategies in terms of the time and cost for each step of the SELEX process, including affinity selection, amplification, and oligonucleotide conditioning. Target-binding oligonucleotides in the enriched pools are sequenced and compared to assess the relative efficacy of the SELEX strategies. We show that the microfluidic strategies are more time- and cost-efficient than conventional SELEX. Aptamers are an alternative category of affinity probes that are much smaller in size, making them ideal probes for intracellular targets. However, few aptamers are developed against intracellular targets, and the few intracellular-targeting aptamers are mainly used as intramers engineered to be expressed inside the target cells, which are unsuitable for intracellular biodosimetry applications. Herein, we use a radiation biomarker BAX as a target, and present an intracellular aptamer developed via microfluidic technologies. The isolated BAX aptamer would allow for in situ labeling of intracellular BAX protein, and we have preliminarily demonstrated the dose-dependent labeling in ex vivo human blood samples. This method could enable the development of aptamers for a panel of intracellular proteins towards radiation biodosimetry applications. Aptamer development involves a screening process following the sequencing of the enriched pool. This process would usually be performed with affinity determination methods, which are often time-consuming and may hinder the development of aptamers. Herein, we reported a graphene-based nanosensor designed for the aptamer screening process. Screening of enriched pool against IgE protein was performed on this sensor. By comparative validation, this sensor showed the capacity to identify the strong binders in the enriched aptamer candidate pools and can be used to expedite aptamer development. Molecular beacons are single-stranded oligonucleotides that adopt a stem-loop structure for in situ hybridization. They can be designed to target radiation-responsive mRNAs, which is a class of biomarkers that are attractive for biodosimetry. We design and use molecular beacons as probes for the measurement of radiation-induced changes of intracellular mRNA in a microfluidic device for the determination of radiation dosage. Our experiments, in which fixed TK6 cells labeled with a molecular beacon specific to BAX mRNA exhibited dose-dependent fluorescence in a manner consistent with RT-qPCR analysis, demonstrate the potential utility of this approach in point-of-care biodosimetry. Molecular beacons against FDXR mRNA have also been developed preliminarily. In summary, this thesis presents the development of multiple molecular probes for intracellular targets, aiming to be applied towards biodosimetry applications. Opportunities for future research are discussed at the end of the thesis, including enhancements in microfluidic measurements of intracellular biomarkers, the development of nucleic acid probes for multiplexed measurements, and the creation of integrated microfluidic devices for point-of-care intracellular biodosimetry.

The initial goal of the project was to develop a more effective method for the purifications of oligonucleotides. This was achieved by developing a method of ion-exchange based on-column deprotection. The resulting method was compared to preestablished methods using LC-MS fraction analysis evaluated according to a purityscore with the acceptance criteria of ≥ 0.45. The on-column deprotection was shownto achieve a purity score > 0.6 for both of the fully thiolated and the partially thiolatedoligonucleotides while the other methods of regular ion-exchange chromatography,ion-pair chromatography and reversed phase chromatography only achieved a purityscore of ≥ 0.6 for only the fully thiolated oligonucleotides. The ion-exchange methodsbecame dependent on more chaotropic agents than expected, but the results gainedduring the resin screening portion of the project may provide a remedy. During theresin screening, the effects of two factors, pore size and ligand density, were investigated. Showing that the pore size did not affect the oligonucleotides to any extent,resulting in the ligand density being the major affecting factor. When studying thedifferent resins, a trend of lower retention with lower ligand density was observed.These results provide a foundation to use lower ligand density resins compatible withless harsh conditions when applying the on-column deprotection in future optimization for the purification of pharmaceutical oligonucleotides. / Syftet med projektet var att utveckla en mer effektiv metod för upprening av oligonukleotider. Detta uppnåddes genom att utveckla en metod för direkt avlägsning av denskyddande gruppen direkt på kolonnen baserat på jonbytes kromatografi. Den resulterande metoden jämfördes med tidigare etablerade metoder genom LC-MS fraktionsanalys och utvärderades enligt ett renhetsbetyg med acceptanskriterier på ≥ 0,45.Metoden visade sig uppnå ett renhetsbetyg > 0,6 för både de fullständigt tiolieradeoch delvis tiolierade oligonukleotider, medan andra metoder som vanlig jonutbyteskromatografi, jonpars-kromatografi och reversed phase-kromatografi endast uppnådde ett renhetsbetyg ≤ 0,6 för fullständigt tiolierade oligonukleotider. Jonutbytesmetoderna blev beroende av fler kaotropiska kemikalier än förväntat, men resultatenfrån resin undersökningen under projektet kan ge en lösning på detta. Under resinundersökningen utreddes effekterna av två faktorer, porstorlek och liganddensitet. Detvisades att porstorleken inte påverkade oligonukleotiderna i någon större utsträckningoch att liganddensiteten var den avgörande faktorn. Vid studier av olika resiner observerades en trend mot lägre retention vid lägre liganddensitet. Dessa resultat ger engrund för att använda resiner med lägre liganddensitet i den utevecklade metoden.Vilket bör tillåta mindre hårda förhållanden vid fortsatt optimering av uppreningenför farmaceutiska oligonucleotider.

A PEGlação é uma bem sucedida estratégia nano-biotecnológica que envolve a ligação covalente do polietilenoglicol (PEG) a uma droga para melhorar sua farmacocinética, farmacodinâmica e perfil imunológico, e portanto, aumentar seu efeito terapêutico. Atualmente, a PEGlação é usada para modificar proteínas, peptídeos, oligonucleotídeos e fragmentos de anticorpos. A Uricase (EC 1.7.3.3, UC) é uma enzima pertencente à classe das oxidorredutases, responsável pela oxidação do ácido úrico, produzindo alantoína. Essa enzima é encontrada em muitos organismos vivos como: bactérias, leveduras, fungos, vegetais e animais. Entretanto, durante a evolução das espécies o gene da UC tornou-se inativo, por isso, em humanos a UC é inativa. Nesse sentido, a UC adquiriu destaque como um potencial fármaco uricolítico, devido à necessidade do desenvolvimento de novos agentes terapêuticos no tratamento de hiperuricemia e gota. Neste estudo, a uricase recombinante purificada de Candida sp (UC-r) e a de rim bovino (UC-b) foram modificadas por PEGlação com mPEG-p-nitrofenil carbonato (mPEG-pNP) e 2-O-mPEG-4,6-dicloro-s-triazina (mPEG-CN), produzindo conjugados com considerável atividade enzimática residual UC-r-mPEG-pNP (87%), UC-r-mPEG-CN (75%) e UC-b-mPEG-pNP (75%), UC-b-mPEG-CN (50%).Além disso, os conjugados obtidos com a UC-r e UC-b apresentaram valores de KM menores do que as enzimas nativas, indicando que a PEGlação conferiu uma interessante propriedade aos conjugados, que permitiu um aumento da afinidade da UC-r e UC-b pelo ácido úrico. O efeito do pH e da temperatura sobre a UC-r e UC-b modificadas indicou que os conjugados obtidos foram mais ativos em pH próximo ao fisiológico e mais estáveis do que a respectiva enzima nativa. As formas PEGladas da UC-r e UC-b foram mais resistentes à ação de diferentes proteases e mantiveram-se estáveis em soro humano, indicando que a PEGlação favoreceu a resistência a degradação proteolítica. Análises espectroscópicas de dicroísmo circular (CD) e infravermelho (FTIR) não apresentaram nenhuma diferença relevante entre a estrutura protéica da UC-r nativa e PEGlada. Estudos in vivo com coelho e camundongos Balb/c mostraram que a UC-r nativa induziu uma intensa resposta imune sendo altamente imunogênica. Por outro lado, a UC-r PEGlada quando injetada cronicamente em camundongos não induziu qualquer resposta detectável de anticorpos. Esses resultados indicam uma suficiente redução da imunogenicidade dessa enzima, devido à conjugação do mPEG-pNP ou mPEG-CN, tornando-a adequada para um possível uso terapêutico. Portanto, nesse trabalho, os resultados obtidos com a UC-r de Candida sp, mostram que dois conjugados apresentaram interessantes propriedades físico-química, biológicas e imunológicas, que permitiram um significativo avanço na transformação de uma enzima de origem fúngica em uma droga, com uma possível aplicação terapêutica no tratamento de hiperuricemia e gota. / PEGylation is a successful nanobiotechnology strategy that involves the covalent attachment of polyethylene glycol (PEG) to a drug to improve its pharmacokinetic, pharmacodynamic, and immunological profiles, and thus, enhance its therapeutic effect. Currently, PEGylation is used to modify proteins, peptides, oligonucleotides, antibody fragments, and small organic molecules. Uricase (EC 1.7.3.3, UC) is an enzyme belonging to the class of oxidorreductases responsible for the oxidation of uric acid, producing allantoin. This enzyme is found in many living organisms such as bacteria, yeasts, fungi, plants and animals. However, during the evolution of the species gene became inactive UC, therefore, in humans UC is inactive. Accordingly, UC has acquired prominence as a potential drug uricolytic due to the need of developing new therapeutic agents for the treatment of hyperuricemia and gout. In this study, purified recombinant uricase from Candida sp (UC-r) and ox kidney (UC-b) were modified by PEGylation with mPEG-p-nitrophenyl-carbonate (mPEG-pNP) and 2-O-mPEG-4,6-dichloro-s-triazine (mPEG-CN), producing conjugates with considerable residual enzyme activity UC-r-mPEG-pNP (87%), UC-r-mPEG-CN (75%) and UC-b-mPEG-pNP (75%),UC-b-mPEG-CN (50%). In addition, conjugates obtained with the UC-r and UC-b had lower KM values than native enzymes, indicating that the PEGylation gave an interesting property the conjugate that increased the affinity of UC-r and UC-b by uric acid. The effect of pH and temperature on the modified UC-r and UC-b indicated that the conjugates were more active at pH close to the physiological and more stable than its native enzyme. PEGylated forms of UC-r and UC-b were more resistant to the action of different proteases and remained stable in human serum, indicating that the PEGylation favored resistance to proteolytic degradation. Spectroscopic analysis of circular dichroism (CD) and infrared (FTIR) did not show any relevant difference in protein structure between native and PEGylated UC-r. In vivo studies with rabbit and Balb/c mice showed that UC-r native elicited an intense immune response being highly immunogenic. On the other hand, the PEGlated UC-r when chronically injected into mice did not induce any detectable response to antibodies. These results indicate a sufficient reduction of immunogenicity of this enzyme, due to conjugation of mPEG-pNP or mPEG-CN, making it suitable for possible therapeutic use. Therefore, the results obtained with the UC-r of Candida sp, showed that two conjugates have interesting physical-chemical, biological and immunological, which allowed a significant advance in the transformation of an enzyme of fungal origin in a drug with a possible application therapeutic in the treatment of hyperuricemia and gout.

by Chan Ka Kui. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 104-113). / Abstracts in English and Chinese. / ABSTRACT --- p.1 / Chapter 1 --- INTRODUCTION --- p.5 / Chapter 1.1 --- Incidence rate of breast cancer in Hong Kong --- p.5 / Chapter 1.2 --- Estrogen and breast cancer --- p.6 / Chapter 1.3 --- The relation between glucose transporters and breast cancer --- p.7 / Chapter 1.4 --- Antisense oligonucleotide --- p.10 / Chapter 1.5 --- Action mechanisms of antisense oligonucleotide --- p.11 / Chapter 1.6 --- Modification of the oligonucleotide --- p.13 / Chapter 1.7 --- Length --- p.16 / Chapter 1.8 --- Sequence selection of the antisense oligonucleotide --- p.16 / Chapter 1.9 --- Delivery means in antisense oligonucleotide --- p.18 / Chapter 1.10 --- The therapeutic role of antisense oligonucleotide --- p.19 / Chapter 1.11 --- Objective of the project --- p.21 / Chapter 2 --- MATERIAL AND METHODS --- p.23 / Chapter 2.1 --- Materials --- p.23 / Chapter 2.2 --- Methods --- p.26 / Chapter 3 --- RESULTS --- p.37 / Chapter 3.1 --- The characteristics of MCF-7 and MDA-MB-231 cells --- p.37 / Chapter 3.2 --- Trend of uptake of antisense oligonucleotides in MCF-7 and MDA- MB-231 cells --- p.41 / Chapter 3.3 --- The integrity of the oligonucleotide in serum-free medium during transfection --- p.48 / Chapter 3.4 --- Detection of effects of Glut5 antisense oligonucleotides of breast tumor cells-MTT assay --- p.50 / Chapter 3.5 --- Detection of the antiproliferative effect by trypan blue exclusion assay and thymidine incorporation --- p.56 / Chapter 3.6 --- Cell cycle analysis and DNA extraction --- p.61 / Chapter 3.7 --- Suppression of Glut5 mRNA detected by RT-PCR --- p.66 / Chapter 3.8 --- Suppression of translation of Glut5 proteins as indicated by Western blotting --- p.73 / Chapter 3.9 --- Measurement of the fructose and glucose uptake in MCF-7 and MDA -MB-231 cells after antisense treatment --- p.76 / Chapter 3.10 --- Change of the phosphofructokinase-1 (PFK-1) activities in MDA- MB-231 cells --- p.82 / Chapter 3.11 --- Measurement of the change in the intracellular pH of the breast tumor cells --- p.84 / Chapter 4 --- DISCUSSION --- p.89 / Chapter 4.1 --- The insights of Glut5 antisense oligonucleotide into cancer therapy --- p.89 / Chapter 4.2 --- The uptake pattern of Glut5 antisense oligonucleotides in breast tumor cells --- p.90 / Chapter 4.3 --- Stability of antisense oligonucleotide during transfection --- p.92 / Chapter 4.4 --- Effects of Glut5 antisense oligonucleotide on MCF-7 and MDA-MB- 231cells --- p.93 / Chapter 4.5 --- Proofs of undergoing antisense action mechanism --- p.95 / Chapter 4.6 --- Physiological changes in breast tumor cells after antisense treatment --- p.97 / Chapter 5 --- CONCLUSION --- p.103 / Chapter 6 --- References --- p.104

Le marché du diagnostic connait un essor considérable depuis l’avènement de labiologie moléculaire. Plus précis et souvent plus rapide, le diagnostic moléculaire in vitro(DIV) est de plus en plus utilisé dans les laboratoires d’analyses médicales. L’ensemble destests dédiés au marché du DIV répond à des contraintes socio-économiques très précisescomme : la fiabilité du résultat, le délai de réponse court, le faible coût et la facilitéd’utilisation. Les indicateurs socio-économiques montrent que la technologie des biopuces estun potentiel bon candidat pour répondre aux attentes du marché. En effet, cet outil permetl’analyse simultanée de plusieurs dizaines voire centaines de séquences nucléiques et doncl’identification d’autant d’organismes en une seule analyse. Cette technologie s’inscrit encomplément de la PCR en apportant l’avantage de l’analyse multiplexée à moyen débit. Deplus, elle permet de donner une réponse globale de la multiplicité des espèces présentes dansl’échantillon sans avoir besoin de passer par une étape de culture. Néanmoins, cettetechnologie n’est pas optimisée pour le marché du DIV. En effet, son usage est complexe, peurobuste et trop cher pour concurrencer les méthodes actuelles (microbiologie pasteurienne,PCR, Elisa, etc..). Dans le but de réduire le coût de fabrication des biopuces à ADN, il estdonc nécessaire de développer des méthodes alternatives. Dans un premier temps, l’objectif de cette thèse Cifre a été de mettre au point unprototype nouveau de dépôt de biomolécules basé sur la lithographie douce, permettant dedéposer les oligonucléotides sondes de façon multiplexée et selon des motifs micrométriques.Cette nouvelle technologie a été évaluée par rapport aux technologies de références. Puis,nous avons développé un procédé innovant de double fonctionnalisation de surface. Ceprocédé simple et rapide a pour avantages de fonctionnaliser la biopuce avec la chimie desurface et les sondes en une seule étape et d’augmenter les signaux d’hybridation. La secondepartie de la thèse a été de coupler cette nouvelle technologie à la détection des événementsd’hybridation sans marquage en utilisant la diffraction de la lumière. La principale différenceavec la méthode de détection par fluorescence repose sur l’adressage des sondes. En effet, ledépôt doit être réalisé sous forme de réseaux de lignes nanométriques diffractants de façon àce que l'interaction entre les molécules déposées et les cibles qui interagissent soit trèssensible. Cette seconde phase du projet a été très ambitieuse et innovante. La faisabilité decette méthode de détection, démontrée par des simulations théoriques, a fait l’objet d’untravail d’optimisation très important et les résultats obtenus montrent que cette technologiesans marquage est possible. / The diagnosis market increased since the advent of molecular biology. More precise and often faster, the in vitro molecular diagnosis (DIV) is more and more used in medical analyses laboratories. DNA chips technology seems to be a good candidate to answer the market expectations. Indeed, this tool allows making several hundreds of analyses simultaneously. Furthermore, it allows giving a global answer of all the present species in the sample without the need of a culture step. Nevertheless, this technology is not optimized for the market of the DIV. Indeed, its use is complex and too expensive in comparison with the current methods (Pasteur microbiology, PCR, Elisa, etc.). So it is necessary to develop an alternative method to reduce the manufacturing cost and simplify the use of DNA chips. First, the goal of this industrial PhD Cifre supported by the Dendris Company was to complete a new prototype of biomolecules deposition based on soft lithography, allowing multiplexing the deposition of oligonucleotides probes along micro and nanometric patterns.This new technology was compared with the reference technologies. Then, we developed an innovative process of surface co-functionalization. This simple and fast process permits to functionalize the DNA chips with both surface chemistry and probes in a single step and to increase the hybridization signals. The second part of this PhD thesis was to couple this new technology with label-free detection using light diffraction. The main difference with fluorescence-based detection was about probes patterning. Indeed, we needed to generate molecular gratings of nanometric lines to diffract efficiently light from a laser beam. We showed that the absolute diffraction intensity increase with the gratings thickness, which is directly correlated with, probes and targets interactions. The second phase of the project was very ambitious and innovative because we demonstrated the feasibility of this label-free detection. And now we can think that this technology will appear as an alternative method for the diagnosis

Lam Mei Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 172-181). / Abstracts in English and Chinese. / Abstract --- p.i / 論文撮要 --- p.iv / Acknowledgement --- p.vii / Table of contents --- p.viii / List of tables --- p.xi / List of figures --- p.xii / Abbreviations --- p.xvii / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- The facilitative glucose transporter family --- p.2 / Chapter 1.2 --- Overexpression of glucose transporters in tumor cells --- p.5 / Chapter 1.3 --- Antisense strategy --- p.8 / Chapter 1.3.1 --- Modifications of oligonucleotides --- p.9 / Chapter 1.3.2 --- Delivery system for oligonucleotides --- p.13 / Chapter 1.3.3 --- Factors influencing antisense activity --- p.16 / Chapter 1.3.4 --- Mechanism of action of antisense oligonucleotides --- p.17 / Chapter 1.3.5 --- Clinical trials of antisense treatment --- p.21 / Chapter 1.4 --- Objective of present study --- p.23 / Chapter Chapter 2: --- Materials and Methods --- p.24 / Chapter 2.1 --- Materials --- p.25 / Chapter 2.1.1 --- Reagents and buffers --- p.25 / Chapter 2.1.2 --- Reagents for Western blot analysis --- p.26 / Chapter 2.1.3 --- Culture medium --- p.28 / Chapter 2.1.4 --- Chemicals --- p.29 / Chapter 2.1.5 --- Culture of cells --- p.31 / Chapter 2.1.5.1 --- Differentiated Human Hepatoblastoma cell line (HepG2) --- p.31 / Chapter 2.1.5.2 --- "Multi-drug resistant hepatoma cell line, R-HepG2 cells" --- p.32 / Chapter 2.1.6 --- Animal Studies --- p.33 / Chapter 2.2 --- Methods --- p.34 / Chapter 2.2.1 --- In vitro studies --- p.34 / Chapter 2.2.1.1 --- Design of oligonucleotide sequence --- p.34 / Chapter 2.2.1.2 --- Transfection --- p.35 / Chapter 2.2.1.3 --- MTT assay --- p.36 / Chapter 2.2.1.4 --- Flow cytometry --- p.37 / Chapter 2.2.1.5 --- H-thymidine incorporation assay --- p.45 / Chapter 2.2.1.6 --- 2-Deoxy-D-[l-3H] glucose uptake assay --- p.46 / Chapter 2.2.1.7 --- Adenosine-5'-triphosphate (ATP) assay --- p.47 / Chapter 2.2.1.8 --- Western blot analysis --- p.50 / Chapter 2.2.2 --- In vivo studies --- p.55 / Chapter 2.2.2.1 --- Animal studies --- p.55 / Chapter (i) --- Lactate dehydrogenase (LDH) assay --- p.58 / Chapter (ii) --- Creatine kinase (CK) assay --- p.60 / Chapter (iii) --- Aspartate transaminase (AST) assay --- p.62 / Chapter (iv) --- Alanine transaminase (ALT) assay --- p.64 / Chapter Chapter 3: --- Results --- p.67 / Chapter 3.1 --- In vitro studies --- p.68 / Chapter 3.1.1 --- Characteristics of the multi-drug resistant cell line (R-HepG2) developed in our laboratory --- p.68 / Chapter 3.1.2 --- Effect of lipofectin on cell viability --- p.77 / Chapter 3.1.3 --- Cellular uptake of antisense oligonucleotide --- p.82 / Chapter 3.1.4 --- Effect of Glut 2 antisense oligonucleotides on human hepatoma HepG2 and its multidrug resistant (R-HepG2) cells by MTT assay --- p.87 / Chapter 3.1.5 --- Suppression of Glut 2 protein expression by antisense oligonucleotides as revealed by Western blot analysis --- p.96 / Chapter 3.1.6 --- Uptake of glucose in HepG2 and R-HepG2 after Glut 2 antisense treatment --- p.100 / Chapter 3.1.7 --- ATP content in HepG2 and R-HepG2 was lowered after treating the cells with antisense oligonucleotides --- p.108 / Chapter 3.1.8 --- Antisense oligonucleotides against Glut 2 exhibited antiproliferative effect on HepG2 and R-HepG2 cells --- p.117 / Chapter 3.1.9 --- Change in cell cycle pattern after antisense treatment --- p.125 / Chapter 3.1.10 --- Glut 2 antisense oligonucleotides did not induce apoptosis --- p.131 / Chapter 3.2 --- In vivo studies --- p.135 / Chapter 3.2.1 --- Effect of antisense oligonucleotides on the tumor weight in nude mice bearing HepG2 cells or R-HepG2 cells --- p.135 / Chapter 3.2.2 --- Assessment of any side effect of antisense drug done on normal tissues of nude mice --- p.139 / Chapter 3.2.2.1 --- Treatment on tumor bearing nude mice with Glut 2 antisense or sense oligonucleotides did not cause myocardial injury --- p.139 / Chapter 3.2.2.2 --- Liver injury was not detected in Glut 2 antisense or sense oligonucleotides treated tumor bearing nude mice --- p.147 / Chapter Chapter 4: --- Discussion --- p.151 / Chapter 4.1 --- In vitro study of the effect of antisense oligonucleotides against Glut 2 on HepG2 and its multi-drug resistant R-HepG2 cell lines --- p.152 / Chapter 4.1.1 --- Design of antisense oligonucleotides against Glut 2 --- p.154 / Chapter 4.1.2 --- Conditions for antisense inhibition by oligonucleotides --- p.155 / Chapter 4.1.3 --- Biological effects of antisense oligonucleotides --- p.158 / Chapter 4.2 --- In vivo study of the effect of antisense oligonucleotides against Glut 2 on HepG2 or R-HepG2 cells bearing nude mice --- p.166 / Chapter 4.2.1 --- Effect of Glut 2 antisense oligonucleotides on tumor weight --- p.167 / Chapter 4.2.2 --- In vivo side effects of oligonucleotides --- p.168 / Chapter 4.3 --- Conclusion --- p.169 / Bibliography --- p.172

<p>Synthetic and physicochemical studies on appropriately functionalized ODN-conjugates have been performed to evaluate their abilities to act as antisense agents against RNA or as intramolecular DNA cross-linking agents. Intercalating aromatic systems [phenazine (Pnz), dipyridophenazine (DPPZ)] and metallointercalators such as Ru²⁺(phen)₂(DPPZ) and Ru²⁺(tpy)(DPPZ)<b>L</b> [where <b>L</b> = chemically or photochemically labile ligand, phen = phenanthroline, tpy = terpyridine], which are covalently tethered to the oligo-deoxynucleotides (ODNs), have been chosen for this purpose. The ODN-conjugates were typically prepared by automated solid phase synthesis using phosphoramidite building blocks, or on solid supports, both functionalized with the chromophore groups. The photosensitive metal complex, Ru²⁺(tpy)(DPPZ)(CH₃CN), has been incorporated by post-synthetic coupling to the amino-linker modified ODNs <i>via</i> an amide bond. The intercalating ability of the tethered chromophores gave enhanced stability of the duplexes and triplexes formed with ODN-conjugates and their complementary targets: DNA, RNA, or double-stranded DNA. The conjugation of DPPZ chromophore to ODN (at 3', 5' or at the middle) led us to incorporate Ru²⁺(phen)₂(DPPZ) through the DPPZ ligand, for the first time. The corresponding (Ru²⁺-ODN)•DNA duplexes showed dramatic stabilization (ΔT_m = 19.4 – 22.0ºC). The CD and DNase I footprinting experiments suggest that the stabilization is owing to metallointercalation by threading of the Ru²⁺(phen)₂ moiety through the ODN•DNA duplex core, thus "stapling" the two helical strands from the minor to major groove. On the other hand, Ru²⁺(tpy)(DPPZ)(CH₃CN)-ODN conjugates represent a new class of oligonucleotides containing the photoactivatible Ru²⁺ complexes, which can successfully crosslink to the complementary strand. The mechanism of cross-linking upon photoirradiation of [Ru²⁺(tpy)(DPPZ)(CH₃CN)-ODN]•DNA involves <i>in situ</i> conversion to the reactive [Ru²⁺(tpy)(DPPZ)(H₂O)-ODN]•DNA which are subsequently cross-linked through the G residue of the complementary DNA strand. All starting materials and products have been purified by HPLC and/or by PAGE and subsequently characterized by MALDI-TOF as well as ESI mass spectroscopy. Terminal conjugation of the planar Pnz and DPPZ groups through the flexible linkers were also shown to improve thermal stability of the ODN•RNA hybrid duplexes without alteration of the initial AB-type global helical structure as revealed from CD experiments. As a result, RNase H mediated cleavage of the RNA strand in the intercalator-tethered ODN•RNA duplexes was more efficient compared to the natural counterpart. The RNase H cleavage pattern was also found to be dependent on the chemical nature of the chromophore. It appeared that introduction of a tether at the 3'-end of the ODN can be most easily tolerated by the enzyme regardless of the nature of the appending chromophore. The tethered DPPZ group has also been shown to chelate Cu²⁺ and Fe³⁺, like phenanthroline group, followed by the formation of redox-active metal complex which cleaves the complementary DNA strand in a sequence-specific manner. This shows that the choice of appropriate ligand is useful to (i) attain improved intercalation giving Tm enhancement, and (ii) sequence-specifically inactivate target RNA or DNA molecules using multiple modes of chemistry (RNase H mediated cleavage, free-radical, oxidative pathways or photocross-linkage).</p>

<p>Antisense oligonucleotides (ODN) may interfere in gene expression on the basis of hybridising to its complementary messenger RNA (mRNA) sequence in the cell thereby preventing the synthesis of the peptide. Therefore, these ODNs may be potential drugs to treat human diseases by “knocking down” the expression of responsible genes or correcting the maturation process of mRNA in the field called antisense therapy. Moreover, antisense ODNs upon labelling are also potential imaging agents to monitor gene expression <i>in vivo</i>, i.e. to accomplish <i>in vivo</i> hybridisation. This would provide a non-invasive tool compared to present methods, which require tissue samples. </p><p>This goal may be reached using positron emission tomography (PET) methodology. PET is a most advanced <i>in vivo</i> imaging technology, which would allow exploring the fate of radionuclide-labelled antisense ODNs in the body; thereby providing information about biodistribution and quantitative accumulation in tissues to assess pharmacokinetic properties of ODNs. This kind of evaluation is important as part of the characterisation of antisense therapeutics but also as part of the development of antisense imaging agents.</p><p>The present study aimed to investigate ⁷⁶Br- and ⁶⁸Ga-labelled ODNs of five different modifications: phosphodiester, phosphorothioate, 2'-<i>O</i>-methyl phosphodiester, locked nucleic acid (LNA), and peptide nucleic acid. The study included exploration of the hybridisation abilities of these ODNs after labelling; furthermore, the biodistribution, metabolite analysis and uptake of the ODNs in rats regarding non-hybridisation and hybridisation specific uptake was conducted. Among the ODNs studied, LNA-DNA mixmer (LNA and DNA nucleotides in alternation along the sequence) displayed the most promising characteristics considering a higher retention in tissues, stability and longer plasma residence. However, biodistribution data demonstrated a non-hybridisation specific distribution in rat tissues with kidney, liver, spleen and bone marrow being the organs of high uptake. Scavenger receptors or other saturable processes unrelated to hybridisation may play a role in tissue uptake and in clearance of antisense ODNs through these organs. These processes may be sequence dependent suggesting that proof of <i>in vivo</i> hybridisation through imaging needs much more elaborate evaluations than just comparison of sense and antisense sequences and proving dose-dependency.</p>

Synthetic and physicochemical studies on appropriately functionalized ODN-conjugates have been performed to evaluate their abilities to act as antisense agents against RNA or as intramolecular DNA cross-linking agents. Intercalating aromatic systems [phenazine (Pnz), dipyridophenazine (DPPZ)] and metallointercalators such as Ru2+(phen)2(DPPZ) and Ru2+(tpy)(DPPZ)<b>L</b> [where <b>L</b> = chemically or photochemically labile ligand, phen = phenanthroline, tpy = terpyridine], which are covalently tethered to the oligo-deoxynucleotides (ODNs), have been chosen for this purpose. The ODN-conjugates were typically prepared by automated solid phase synthesis using phosphoramidite building blocks, or on solid supports, both functionalized with the chromophore groups. The photosensitive metal complex, Ru2+(tpy)(DPPZ)(CH3CN), has been incorporated by post-synthetic coupling to the amino-linker modified ODNs via an amide bond. The intercalating ability of the tethered chromophores gave enhanced stability of the duplexes and triplexes formed with ODN-conjugates and their complementary targets: DNA, RNA, or double-stranded DNA. The conjugation of DPPZ chromophore to ODN (at 3', 5' or at the middle) led us to incorporate Ru2+(phen)2(DPPZ) through the DPPZ ligand, for the first time. The corresponding (Ru2+-ODN)•DNA duplexes showed dramatic stabilization (ΔTm = 19.4 – 22.0ºC). The CD and DNase I footprinting experiments suggest that the stabilization is owing to metallointercalation by threading of the Ru2+(phen)2 moiety through the ODN•DNA duplex core, thus "stapling" the two helical strands from the minor to major groove. On the other hand, Ru2+(tpy)(DPPZ)(CH3CN)-ODN conjugates represent a new class of oligonucleotides containing the photoactivatible Ru2+ complexes, which can successfully crosslink to the complementary strand. The mechanism of cross-linking upon photoirradiation of [Ru2+(tpy)(DPPZ)(CH3CN)-ODN]•DNA involves in situ conversion to the reactive [Ru2+(tpy)(DPPZ)(H2O)-ODN]•DNA which are subsequently cross-linked through the G residue of the complementary DNA strand. All starting materials and products have been purified by HPLC and/or by PAGE and subsequently characterized by MALDI-TOF as well as ESI mass spectroscopy. Terminal conjugation of the planar Pnz and DPPZ groups through the flexible linkers were also shown to improve thermal stability of the ODN•RNA hybrid duplexes without alteration of the initial AB-type global helical structure as revealed from CD experiments. As a result, RNase H mediated cleavage of the RNA strand in the intercalator-tethered ODN•RNA duplexes was more efficient compared to the natural counterpart. The RNase H cleavage pattern was also found to be dependent on the chemical nature of the chromophore. It appeared that introduction of a tether at the 3'-end of the ODN can be most easily tolerated by the enzyme regardless of the nature of the appending chromophore. The tethered DPPZ group has also been shown to chelate Cu2+ and Fe3+, like phenanthroline group, followed by the formation of redox-active metal complex which cleaves the complementary DNA strand in a sequence-specific manner. This shows that the choice of appropriate ligand is useful to (i) attain improved intercalation giving Tm enhancement, and (ii) sequence-specifically inactivate target RNA or DNA molecules using multiple modes of chemistry (RNase H mediated cleavage, free-radical, oxidative pathways or photocross-linkage).

Antisense oligonucleotides (ODN) may interfere in gene expression on the basis of hybridising to its complementary messenger RNA (mRNA) sequence in the cell thereby preventing the synthesis of the peptide. Therefore, these ODNs may be potential drugs to treat human diseases by “knocking down” the expression of responsible genes or correcting the maturation process of mRNA in the field called antisense therapy. Moreover, antisense ODNs upon labelling are also potential imaging agents to monitor gene expression in vivo, i.e. to accomplish in vivo hybridisation. This would provide a non-invasive tool compared to present methods, which require tissue samples. This goal may be reached using positron emission tomography (PET) methodology. PET is a most advanced in vivo imaging technology, which would allow exploring the fate of radionuclide-labelled antisense ODNs in the body; thereby providing information about biodistribution and quantitative accumulation in tissues to assess pharmacokinetic properties of ODNs. This kind of evaluation is important as part of the characterisation of antisense therapeutics but also as part of the development of antisense imaging agents. The present study aimed to investigate 76Br- and 68Ga-labelled ODNs of five different modifications: phosphodiester, phosphorothioate, 2'-O-methyl phosphodiester, locked nucleic acid (LNA), and peptide nucleic acid. The study included exploration of the hybridisation abilities of these ODNs after labelling; furthermore, the biodistribution, metabolite analysis and uptake of the ODNs in rats regarding non-hybridisation and hybridisation specific uptake was conducted. Among the ODNs studied, LNA-DNA mixmer (LNA and DNA nucleotides in alternation along the sequence) displayed the most promising characteristics considering a higher retention in tissues, stability and longer plasma residence. However, biodistribution data demonstrated a non-hybridisation specific distribution in rat tissues with kidney, liver, spleen and bone marrow being the organs of high uptake. Scavenger receptors or other saturable processes unrelated to hybridisation may play a role in tissue uptake and in clearance of antisense ODNs through these organs. These processes may be sequence dependent suggesting that proof of in vivo hybridisation through imaging needs much more elaborate evaluations than just comparison of sense and antisense sequences and proving dose-dependency.