Riboswitches are regulatory structures present in the 5′-UTR of a wide range of bacterial mRNAs. They consist of a small-molecule binding aptamer domain, which affects the conformation of a nearby expression platform to control gene expression through a transcriptional or translational mechanism. Because of their ability to bind selectively to very small concentrations of ligand, in a protein-independent manner, they have great potential for use as novel small-molecule controllable gene expression systems. This thesis describes how a combination of chemical genetics and genetic selection were used to develop and test a novel riboswitch-based gene-expression system. Several constructs were generated which could respond in vivo to a variety of non-natural small heterocyclic compounds and output via a simple fluorescence based assay in a dose-dependent manner. Methods for controlling the overall protein expression landscape of the riboswitch-based gene-expression system are outlined. In addition, the rational design of mutant riboswitch aptamers with improved ligand-binding capabilities is described alongside attempts to modulate the structural stability of the expression platform. Riboswitches need to be highly discriminatory to function effectively in vivo, binding to one ligand from a cellular pool of thousands. Mutant riboswitches were created that responded specifically to the ligands ammeline or azacytosine, and were found to have no induction in the presence of adenine, the wild-type riboswitch ligand. This in vivo ligand orthogonality was confirmed by subsequent in vitro studies. The ligand-induced structural changes undertaken by the mutant riboswitch aptamer domains were subsequently characterised using a variety of in vitro methods including SHAPE, ITC and x-ray crystallography. Finally, the feasibility of using riboswitch gene-expression systems in fully synthetic applications was demonstrated through the construction and analysis of small synthetic gene clusters and operons. The in vivo expression of two fluorescent proteins under independent riboswitch control was studied under single and dual induction for a range of ligand concentrations. The ability to control the expression of multiple genes is highly desirable in the emerging field of synthetic biology, the results described here indicate that riboswitches are ideally suited to complement current gene expression tools.

La détection rapide et sensible des agents pathogènes est d’une très grande importance pour la biosécurité. Les biopuces sont bien adaptées à cet effet, car elles permettent la détection multiplexe des cibles. Une limitation cruciale des biopuces est leur manque de fiabilité et de sensibilité. L’objectif de cette thèse est de développer une architecture reproductible de biopuces à base de couche mince de silicium amorphe carboné (a-SiC:H) déposée sur un réflecteur en aluminium pour une détection fiable et sensible des pathogènes. Nous avons choisi comme système modèle l’interaction de la toxine alimentaire ochratoxine A (OTA) avec son aptamère AntiOTA de longueur 36mer. Les aptamères (simples brins d’ADN) sont de plus en plus utilisés comme sondes en raison de leur grande spécificité et affinité vis-à-vis d’une large gamme de cibles (i.e. protéines, bactéries…). La stratégie de fabrication consiste en un greffage de monocouches organiques d’acides carboxyliques via des liaisons Si-C robustes, suivi de l’accrochage covalent des aptamères par un couplage peptidique. Les processus de greffage ont été mis au point sur silicium cristallin permettant la quantification des couches greffées par spectroscopie infrarouge en mode ATR (Attenuated total reflexion). La quantification IR des interactions OTA – AntiOTA a été montrée pour la première fois sur des surfaces par IR-ATR. La spécificité de l’aptamère a été démontrée en utilisant une molécule chimiquement similaire (warfarin), pour laquelle l’AntiOTA ne montre aucune affinité. Ces protocoles bien contrôlés ont été transférés sur l’architecture de la biopuce a-SiC:H. Les aptamères immobilisés sont hybridés avec des brins complémentaires marqués avec des fluorophores. En présence de l’OTA une déshybridation des brins complémentaires est attendue, conduisant à une diminution du signal fluorescent. Différentes longueurs de brins complémentaires ont été comparées, montrant jusqu’à 13% de diminution due à l’interaction de l’OTA. / Rapid and sensitive detection of pathogenic targets play a crucial role in biosecurity. Biochips are ideal for this, as they allow easy and multiplex detection of targets. A crucial limitation in biochips is that they often suffer from low reliability and sensitivity. The goal of this thesis is to develop a stable and reproducible architecture for biochips based on an amorphous silicon carbon alloy (a-SiC:H) deposited on an aluminium back-reflector for reliable and sensitive detection of pathogens. On these biochips we introduced the interaction of the food and feed toxin ochratoxin A (OTA) with its 36mer aptamer AntiOTA as a model system. Aptamers (single strands of DNA) are ideal as probes for biochips as they display high specificity and affinity towards a wide range of targets (i.e. proteins, bacteria…). The well-controlled multi-step fabrication process consists of the reliable photochemical grafting of acid-terminated organic monolayers on silicon surfaces by robust Si C bonds, which in turn were functionalized with aptamers by stable peptide coupling. Carrying out this process on crystalline silicon allowed monitoring and quantification of every step by infrared spectroscopy (IR-ATR). The interaction OTA – AntiOTA was shown for the first time on surfaces by IR, and an IR in situ calibration allowed the quantification of OTA which was bound by the aptamers on the surface. The specificity of AntiOTA towards OTA was demonstrated by using a chemically similar molecule (warfarin), for which AntiOTA shows no affinity. The well-controlled protocols were transferred to the a-SiC:H biochip. The immobilized aptamers were hybridized with complementary and fluorescent-labeled DNA-strands. In presence of OTA, dehybridization of the complementary strands is expected, resulting in a decrease of fluorescent signal. Different lengths of complementary strands were compared, exhibiting up to 13% signal decrease due to OTA.

京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第19415号 / エネ博第323号 / 新制||エネ||65(附属図書館) / 32440 / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 片平 正人, 教授 森井 孝, 教授 木下 正弘 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

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The functionality of nucleic acids beyond genetics has attracted more attention over the past decades. Functional nucleic acids (FNA), including aptamers and nucleic acid-based enzymes, are well-known for their target binding and reaction catalysis abilities. FNA can be obtained through a technology called in vitro selection, which allows the isolation of customized FNA for various applications. In particular, FNA have received much interest in biosensing application. Their wide range of sensing targets, intrinsic stability, and high specificity have qualified them as the molecular recognition element in biosensors. This thesis explored the utilization of FNA to tackle real-world biosensing challenges, especially for pathogenic bacteria detection. The first project aimed to make the most use of in vitro selection to derive FNA that can meet the requirements of terminal applications. A few feasible approaches were proposed based on lessons from Mother Nature and validated by innovative scientist pioneers. In the second research project, I characterized an RNA-cleaving DNAzyme for Clostridium difficile infection diagnosis. This DNAzyme displayed high sensitivity and specificity for clinical C. difficile strains, making it a competitive candidate for a potential point-of-care diagnostic tool. In the next chapter, I incorporated a Legionella pneumophila-responsive RNA-cleaving DNAzyme into a bead-based assay for practical on-site detection. This assay exhibited a high stability and functionality in the cooling tower water samples, the real-world application environment. The following chapter was to optimize this assay further with a coupled rolling circle amplification strategy. This additional amplification speeded up the detection process, improved the limit of detection, and enabled the colorimetric results that are observable to the naked eye. These research aimed to advance the practical applications of FNA as key components in biosensors. I hope readers find this thesis insightful and inspirational for the development of the FNA field. / Thesis / Doctor of Philosophy (PhD)

Nucleic acids are not only one of the most important classes of macromolecules in biochemistry but also a promising platform for the defined arrangement of chromophores. Thanks to their precise organization by directional polar and hydrophobic interactions, oligonucleotides can be exploited as suitable templates for multichromophore assemblies with predictable properties. To expand the toolbox of emissive, base pairing nucleobase analogs several barbituric acid merocyanine (BAM) chromophores with tunable spectroscopic properties were synthesized and incorporated into RNA, DNA and glycol nucleic acid (GNA) oligonucleotides. A multitude of duplexes containing up to ten BAM chromophores was obtained and analysis by spectroscopic methods revealed the presence of dipolarly coupled merocyanine aggregates with properties strongly dependent on the chromophore orientation toward each other and the backbone conformation. These characteristics were exploited for various applications such as FRET pair formation and polymerase chain reaction (PCR) experiments. The observed formation of higher-order aggregates implies future applications of these new oligonucleotide-chromophore systems as light-harvesting DNA nanomaterials. Besides oligonucleotide templated covalent assembly of chromophores also non-covalent nucleic acid-chromophore complexes are a broad field of research. Among these, fluorogenic RNA aptamers are of special interest with the most versatile ones based on derivatives of the GFP chromophore hydroxybenzylidene imidazolone (HBI). Therefore, new HBI-derived chromophores with an expanded conjugated system and an additional exocyclic amino group for an enhanced binding affinity were synthesized and analyzed in complex with the Chili aptamer. Among these, structurally new fluorogenes with strong fluorescence activation upon binding to Chili were identified which are promising for further derivatization and application as color-switching sensor devices for example. / Nukleinsäuren sind nicht nur eine der wichtigsten Klassen biochemisch relevanter Makromoleküle, sondern stellen auch eine vielversprechende Plattform für die definierte räumliche Organisation kleiner funktioneller Moleküle, wie beispielsweise Chromophore, dar. Oligonu-kleotide können aufgrund ihrer präzise gegliederten Struktur, die durch gerichtete polare und hydrophobe Wechselwirkungen hervorgerufen wird, als nützliche Template für die mehrfache kovalente und nicht-kovalente Anordnung von Chromophoren zu Aggregaten mit vorhersagbaren spektroskopischen Eigenschaften genutzt werden. Obwohl eine Vielzahl solcher Chromophorsysteme bereits in der Literatur beschrieben ist, basieren die meisten Chromophordesigns nur auf hydrophoben Wechselwirkungen zwischen den einzelnen Chromophoren und lassen die intrinsische Fähigkeit kanonischer Nukleobasen, komplementäre Basenpaare zu bilden, außen vor. Allerdings liegt es auf der Hand, dass die Berücksichtigung dieser polaren Wechselwirkungen nicht nur zu einer Stabilisierung der Oligonukleotid-Sekundärstruktur führen kann, sondern auch die Interpretation spektroskopischer Effekte vereinfacht. Um das bekannte Spektrum emittierender Nukleobasen-Analoga zu erweitern und den zusätz-lichen Einfluss dieser polaren Wechselwirkungen auszunutzen, wurden im Zuge dieser Arbeit verschiedene, strukturell unterschiedliche Barbitursäure-Merocyanin-Chromophore (BAM) entworfen. Die Barbitursäure-Akzeptoreinheit dieser künstlichen Nukleobasensurrogate ähnelt der Watson-Crick-Basenpaarungsseite der natürlichen T- und U-Nukleobasen und soll somit die Basenpaarung mit Adenosin ermöglichen. Durch die Kombination dieses Akzeptors mit unterschiedlich aufgebauten aromatischen Donoreinheiten, wie zum Beispiel Indol und Benzothiazol, konnten Merocyanine mit interessanten spektroskopischen Eigenschaften erhalten werden. Da die Konstitution des Nukleinsäurerückgrates einen starken Einfluss auf die Strukturparameter und die thermodynamische Stabilität der resultierenden Duplexstruktur hat, wurden die synthetisierten BAM-Chromophore in Phosphoramiditbausteine für die kovalente Assemblierung innerhalb verschiedener Oligonukleotidsysteme umgewandelt. Neben der Synthese entsprechender DNA- und RNA-Nukleotide wurden die BAM-Chromophore auch als Glykolnukleinsäure-Bausteine (GNA) mit einem azyklischen Rückgrat hergestellt. Der erfolgreiche Einbau der erhaltenen künstlichen Nukleoside konnte durch Festphasensynthese erreicht werden, wobei über 100 modifizierte Einzelstränge erhalten werden konnten. Die Hybridisierung der künstlichen Oligonukleotid-Einzelstränge mit ihren jeweiligen Gegensträngen führte zu einer Vielzahl kurzer Duplexstrukturen mit bis zu zehn BAM-Chromophoren in unterschiedlicher Anordnung. Mithilfe verschiedenster spektroskopischer Methoden konnte ein Einblick in die strukturelle Organisation der Merocyanine innerhalb dieser Systeme erhalten werden, wobei sich die Bildung dipolar-gekoppelter Merocyanin-Dimere und -Multimere zeigte. Die hierfür erforderliche ungewöhnliche \textit{syn}-Konformation der BAM-Chromophore wurde weiterhin durch Oligonukleotid-NMR bestätigt. Erstaunlicherweise wiesen die spektroskopischen und thermodynamischen Eigenschaften BAM-modifizierter Nukleinsäuren eine starke Abhängigkeit von der Chromophororientierung und der Konformation des Rückgrates auf. Dieser Effekt konnte für verschiedene Anwendungen wie die Bildung von FRET-Paaren und die Verwendung als internes fluoreszentes Stop-Nukleotid in Polymerasekettenreaktionen ausgenutzt werden. Mithilfe von Rasterkraftmikroskopie wurde außerdem die Bildung von Aggregaten höherer Ordnung beobachtet, was eine zukünftige Verwendung dieser neuen Oligonukleotid-Chromophor-Systeme als Materialien für Lichtsammelkomplexe oder für die DNA-Nanotechnologie denkbar macht. Ein weiteres großes Forschungsfeld neben kovalenten Chromophoranordnungen mit Oligonukleotiden als Templat sind nicht-kovalente Nukleinsäure-Chromophorkomplexe. Insbesondere fluorogene RNA-Aptamere sind hier von großer Bedeutung wobei die wichtigsten auf der Fluoreszenzaktivierung von Derivaten 4-Hydroxybenzylidenimidazolon-Fluorophors (HBI), dem Chromophor des natürlich vorkommenden grün fluoreszierenden Proteins (GFP), beruhen. Allerdings zeigen viele der berichteten Aptamer-Ligand-Systeme signifikante Nachteile wie unter anderem unspezifische Bindung, eine starke Tendenz zu Photoisomerisierung oder ineffiziente Zellpermeabilität. Deshalb wurden im Zuge dieser Arbeit neue, von HBI abgeleitete Chromophore mit einem vergrößerten konjugierten $\uppi$-System und einer zusätzlichen exozykli-schen Aminogruppe für eine erhöhte Bindungsaffinität synthetisiert und im Komplex mit dem bekannten Chili-Aptamer untersucht. Einige dieser strukturell neuen Chromophore zeigten einen starken Anstieg der Emission bei Bindung an dieses Aptamer und wurden daher weiter charakterisiert. Sie stellen eine vielversprechende Möglichkeit für weitere Derivatisierung und die zukünftige Anwendung beispielsweise als schaltbare Aptamer-basierte Fluoreszenzsensoren dar.

COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID 19 is detected by RT-PCR tests and serological tests. RT-PCR tests detect viral RNA and require trained individuals to run the test as well as a lengthy analysis time. Serological tests detect antibodies produced in response to viral infection. Rapid antigen detection (RAD) tests, such as the at-home COVID test kits, are quick and easy to run. RAD tests detect viral antigen in the test sample binding to the antibody-coated testing device. However, production of antibodies is a long and costly process. Aptamers can replace antibodies with advantages including low-cost, stability, tunable selectivity, and ability to be chemically modified. Aptamers are short single-stranded oligonucleotides selected for specific targets using Systematic Evolution of Ligands by Exponential Enrichment (SELEX). This project aims to characterize the binding of aptamers to SARS-CoV-2 nucleocapsid (N) protein using capillary electrophoresis (CE) and compare with bio-layer interferometry (BLI). DNA aptamers were selected via SELEX and screened using BLI in which protein was immobilized on the BLI sensor tip and dipped into aptamer solution. Three aptamers specific to N protein were selected for further binding affinity (Kd) determination. In CE, the aptamer and protein are free in solution to bind and unbind, providing an alternative approach in characterizing the binding. A greater Kd was observed with CE compared to BLI. Using CE, the apparent Kd of the 3 aptamers was determined to be 18 ± 4 nM, 45 ± 11 nM, and 32 ± 7 nM, respectively. When tested with BLI, the apparent Kd were 4.83 ± 0.63, 4.51 ± 0.87, and 2.91 ± 0.59 nM, respectively. This discrepancy in affinity can be due to steric differences between immobilized (BLI) and in solution (CE) binding, buffer composition and stability of aptamer structures, or buffer pH and difference in electrostatic interactions. All three of these variables will impact binding and the calculated Kd. This work offers insight into aptamer affinity when used in a different system from which they were selected. This work would lead to a better understanding when employing aptamers to different assays and assay mediums.

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