Thema dieser Arbeit ist die evolutive Optimierung der T7-RNA-Polymerase. Zur Stabilisierung technischer oder therapeutischer RNA-Moleküle gegenüber RNAsen wäre es wünschenswert eine RNA-Polymerase zu generieren, welche RNA vollständig aus 2’- modifizierten Nucleotiden synthetisieren kann. Zu diesem Zweck wurde ein kombiniertes Selektions- und Screeningverfahren zur Durchmusterung von Variantenbibliotheken der T7- RNA-Polymerase hinsichtlich des Einbaus von 2’-Methoxy-modifizierten Nucleotiden in RNA entwickelt. Es wurden ein gut handhabbarer, cis-regulierter Expressionsvektor sowie ein Selektionsplasmid erzeugt, die zusammen in E. coli ein in-vivo-Selektionssystem bilden, mit dessen Hilfe man Zellen, welche T7-RNA-Polymerase-Aktivität zeigen anhand ihrer grünen Fluoreszenz identifizieren konnte. Durch error-prone PCR wurden Mutantenbibliotheken generiert, und diese in das Selektionssystem eingesetzt. So konnte die Anzahl der potentiell zu testenden Varianten erheblich gesenkt werden. Zur Bestimmung der T7-RNA-Polymerase- Aktivität mit 2’-Methoxy-modifizierten Nucleotiden wurde ein Fluoreszenz-basierendes Assay etabliert. Dieses Assay, das nicht mit radioaktiv-markierten Nucleotiden arbeitete und keinen gelelektrophoretischen Separationsschritt benötigte, konnte in allen Schritten zur parallelen Bearbeitung von 96 Proben in einem Mikrotiterplatten-Format angepasst werden, so dass es prinzipiell hochdurchsatzfähig war und sich zum Screening umfangreicher Variantenbibliotheken eignete. Die Assay-Reaktion kann dabei auch unkompliziert auf ein Screening von RNA- oder DNA-Polymerase-Bibliotheken hinsichtlich anderer Eigenschaften der Polymerase-Aktivität übertragen werden.

Thema dieser Arbeit ist die evolutive Optimierung der T7-RNA-Polymerase. Zur Stabilisierung technischer oder therapeutischer RNA-Moleküle gegenüber RNAsen wäre es wünschenswert eine RNA-Polymerase zu generieren, welche RNA vollständig aus 2’- modifizierten Nucleotiden synthetisieren kann. Zu diesem Zweck wurde ein kombiniertes Selektions- und Screeningverfahren zur Durchmusterung von Variantenbibliotheken der T7- RNA-Polymerase hinsichtlich des Einbaus von 2’-Methoxy-modifizierten Nucleotiden in RNA entwickelt. Es wurden ein gut handhabbarer, cis-regulierter Expressionsvektor sowie ein Selektionsplasmid erzeugt, die zusammen in E. coli ein in-vivo-Selektionssystem bilden, mit dessen Hilfe man Zellen, welche T7-RNA-Polymerase-Aktivität zeigen anhand ihrer grünen Fluoreszenz identifizieren konnte. Durch error-prone PCR wurden Mutantenbibliotheken generiert, und diese in das Selektionssystem eingesetzt. So konnte die Anzahl der potentiell zu testenden Varianten erheblich gesenkt werden. Zur Bestimmung der T7-RNA-Polymerase- Aktivität mit 2’-Methoxy-modifizierten Nucleotiden wurde ein Fluoreszenz-basierendes Assay etabliert. Dieses Assay, das nicht mit radioaktiv-markierten Nucleotiden arbeitete und keinen gelelektrophoretischen Separationsschritt benötigte, konnte in allen Schritten zur parallelen Bearbeitung von 96 Proben in einem Mikrotiterplatten-Format angepasst werden, so dass es prinzipiell hochdurchsatzfähig war und sich zum Screening umfangreicher Variantenbibliotheken eignete. Die Assay-Reaktion kann dabei auch unkompliziert auf ein Screening von RNA- oder DNA-Polymerase-Bibliotheken hinsichtlich anderer Eigenschaften der Polymerase-Aktivität übertragen werden.

Le système d’expression le plus utilisé pour la production des protéines membranaires, est le système basé sur l’ARN polymérase T7 (ARNpol T7) (Hattab et al., 2015). L'inconvénient de ce système est néanmoins que la vitesse de transcription de l’ARNpol T7 est dix fois plus rapide que celle de l’enzyme bactérienne. Depuis l’isolement de mutants spontanés, notamment C41 (DE3) et C43 (DE3) (Miroux et Walker, 1996) et l’identification de leurs mutations dans le génome, il apparaît clairement que la toxicité provoquée par la surproduction des protéines membranaires est liée à la quantité trop élevée d’ARNpol T7 dans la cellule (Wagner et al., 2008 ; Kwon et al., 2015). Les protéines membranaires ont besoin d’une vitesse de transcription/traduction plus basse pour se replier correctement dans la membrane de la bactérie. Le premier objectif de ma thèse était d’étendre l’amplitude du promoteur du système T7 sur laquelle est basée l’expression des protéines. Pour cela, nous avons isolé et caractérisé de nouvelles souches bactériennes dans lesquelles le niveau d’ARNpol T7 était efficacement régulé par un mécanisme non transcriptionnel très favorable à l’expression des protéines membranaires (Angius et al., 2016). Le deuxième objectif était de comprendre la prolifération des membranes intracellulaires chez E. coli suite à la surexpression de la protéine AtpF, une sous unité membranaire du complexe de l’ATP synthétase (Arechaga et al., 2000). Pour mieux comprendre les voies métaboliques impliquées dans la biogenèse, la prolifération et l’organisation des membranes, nous avons utilisé une approche de séquençage d’ARN à haut débit à différents temps après induction de la surexpression de la sous-unité AtpF dans la souche C43 (DE3). Ensuite, et en collaboration avec Gerardo Carranza and Ignacio Arechaga (Université de Cantabria, Espagne), nous avons construit et étudié des mutants de C43 (DE3) déficients pour les trois gènes codants pour des enzymes de la biosynthèse des cardiolipides afin d’évaluer leur participation dans la biogénèse des membranes intracellulaires / The most successful expression system used to produce membrane proteins for structural studies is the one based on the T7 RNA polymerase (T7 RNAP) (Hattab et al., 2015). However, the major drawback of this system is the overtranscription of the target gene due to the T7 RNAP transcription activity that is over ten times faster than the E. coli enzyme. Since the isolation of spontaneous mutants, namely C41(DE3) and C43(DE3) (Miroux and Walker, 1996) and the identification of their mutation in the genome, it becomes clear that reducing the amount of the T7 RNAP level removes the toxicity associated with the expression of some membrane proteins (Wagner et al., 2008; Kwon et al., 2015). Also, some membrane proteins require a very low rate of transcription to be correctly folded at the E. coli membrane. The first objective of my PhD was to extend the promoter strength coverage of the T7 based expression system. We used genetic and genomic approaches to isolate and characterize new bacterial strains (Angius et al., 2016) in which the level of T7 RNAP is differently regulated than in existing hosts. A second objective was to understand intracellular membrane proliferation in E. coli. Indeed it has been shown that over-expression of membrane proteins, like overexpression of AtpF of E. coli F1Fo ATP synthase is accompanied by the proliferation of intracellular membranes enriched in cardiolipids (Arechaga et al., 2000). To understand metabolic pathways involved in membrane biogenesis, proliferation and organization, we used a RNA sequencing approach at several time point upon over-expression of the F-ATPase b subunit in C43(DE3) host. On the other hand, in collaboration with Gerardo Carranza and Ignacio Arechaga (University of Cantabria, Spain) we studied C43(DE3) cls mutants, in which the cardiolipids genes A, B and C are deleted, to test how they participate to intracellular membranes structuration

<p>Borane (BH<sub>3<sub>) chemistry offers unique chemical characteristics that enable boranophosphate (BP) oligonucleotides with potential to enhance RNA therapeutic applications such as RNA interference (RNAi) and RNA aptamers. Further, BP nucleotides are substrates for RNA polymerases which allow the enzymatic synthesis of stereoregular boranophosphate (BP)-RNA molecules of different lengths and properties. We expect that these BP-RNAs will interact in a novel way with the desired target molecules because they can coordinate with a diverse array of ligand sites in proteins or other RNA molecules. This is due to the distinct hydrophobicity, sterospecificity, and polarity properties imparted by the phosphorus-boron (P-B) chemical bond compared to the natural phosphorus-oxygen (P-O) bond. </p><p>The object of this dissertation is to explore the therapeutic applications of the BP-RNA such as siRNA, RNA aptamers, and in addition investigate the immunogenicity of this modification. We used mouse cells to determine if BP-RNA would activate toll-like receptor (TLR 7), which is involved in innate immune response to foreign single stranded RNA (ssRNA). This response is undesired when applied to oligonucleotide therapeutics such as siRNA and RNA aptamers. In terms of RNAi, it would be an advantage to have low immunogenicity and high downregulation activity by the siRNA. To determine the innate immune activation of the BP-RNA through the TLR 7 we used a known activator, the human immunodeficiency virus (HIV) derived single-stranded RNA (ssRNA40) and measured the production of cytokines as a function of the number of modified BP-linkages. The production of cytokines IL-6 and TNF&#945; was quantified after the boranophosphate (BP), phosphorothioate (PS) or natural ssRNA40 were transfected into murine macrophage Raw264.7 cells. Natural and phosphorothioate RNA (PS-RNA) have been shown to be activators of TLR 7 receptors. In contrast, we found that fully modified BP- ssRNA40 did not activate TLR 7. This is relevant in oligonucleotide applications such as siRNA and RNA aptamers where off-target effects such as immune activation after administration are not desired. </p><p>Subsequently, the low immune activation would be an advantage when coupled to RNAi activity of the oligonucleotide. Thus, we explored whether BP modified siRNA molecules would modulate gene expression and if there was an effect on downregulation activity when increasing the number of BH3 modifications on the phosphate backbone. Our therapeutic model was the multi-drug resistance 1 (MDR1) gene that expresses P-glycoprotein (P-gp), which has been notoriously difficult to modulate. The aberrant regulation of genes such as MDR1 in cancer cells are a major cause of chemotherapeutic treatment failure against human cancers. Hence, controlling the expression of cancer genes with antisense technology is a possible cancer therapy. Specifically, correcting the overexpression of p-glycoprotein using modified siRNAs that target and degrade the P-glycoprotein mRNA produced by the MDR1 gene. We found that there is a reduction of siRNA activity with an increasing number of BP-modifications. It appears that there is a fine balance between lack of immune response and gene downregulation when applied to BP-siRNA. </p><p>Finally, we compared the enrichment during the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method of two libraries, one BP-RNA (U&#945;B) compared to a doubly-modified RNA (2'FC & U&#945;B), against a human thrombin. Aptamers modulate protein activity and interfere with protein signaling by binding to the desired protein with high affinity and specificity leading to their use in therapeutic applications where protein activity needs to be controlled or it is anomalous. In the case of blood coagulation, thrombin plays a central role in coagulation signaling cascade and it is a good target to use to control blood coagulation in clinical settings. We attempted to optimize the selection of BP- RNA aptamers through 4-8 rounds of SELEX against the protein thrombin. We found that the selection conditions were not optimal for BP-RNA SELEX possibly due to non-specific binding to a bovine serum albumin (BSA) in the selection buffer.</p> / Dissertation

In this work, we seek to expand synthetic in vitro biological systems by using water-in-oil emulsions to provide an environment conducive to directed evolution. We approach this primarily by utilizing a model transcription system, the T7 RNA polymerase and promoter, which is orthogonal to both bacterial and eukaryotic transcription systems and is highly functional in vitro. First, we develop a method to identify functional promoter sequences completely in vitro. This method is tested using the T7 RNA polymerase-promoter model system. We then configure the T7 transcription system as an ‘autogene’ and investigate how this positive feedback circuit (whereby a T7 promoter expresses a T7 RNA polymerase gene) functions across various in vitro platforms, including while compartmentalized. The T7 autogene can be envisioned as a self-replicating system when compartmentalized, and its use for directed evolution is examined. Finally, we look towards future uses for these in vitro systems. One interesting application is to expand the utilization of unnatural base pairs within the context of a synthetic system. We investigate the ability of T7 RNA polymerase to recognize and utilize unnatural base pairs within the T7 promoter, complementing existing work on the utilization of unnatural base pairs for in vitro replication and transcription with an investigation of more complex protein-dependent regulatory function. We envision this work as a foundation for future in vitro synthetic biology efforts. / text

Da interação da actina com a miosina resulta a contração muscular. Esta interação é controlada pela concentração de íons cálcio, que atuam sobre um sistema regulatório associado ao filamento de actina. O sistema regulatório é composto de uma molécula de tropomiosina e um complexo protéico composto de três polipeptídeos chamado troponina. Uma das subunidades, troponina C, e o receptor de ions cálcio; outra, troponina T, liga-se à tropomiosina; e a terceira, troponina I, é a responsável pela inibição da atividade ATPásica da actomiosina. Esta dissertação descreve o desenvolvimento do processo de expressão da troponina I em bactéria, sua purificação e caracterização. Partindo de um cDNA de troponina I músculo esquelético de galinha, procedemos à construção de um vetor de expressão da proteína em E.coli com o sistema pET. Inicialmente construímos um vetor de expressão capaz de produzir a troponina I fundida a 18 aminoácidos. A proteína foi purificada e caracterizada, comportando-se de forma semelhante à troponina I selvagem. Numa segunda etapa, foram feitas mutações sítio-dirigidas para a construção de um novo sítio de restrição que possibilitou construir um vetor de expressão da troponina I a partir de sua metionina inicial. Entretanto não foi obtida expressão com esta construção. Para solucionar o problema, foram feitas novas mutações que alteraram os codons AGG presentes nos 25 primeiros codons do gene da proteína por codons CGT, sem alterar o aminoácido codificado. Nesta construção final foi obtida a expressão da proteína sem fusão, que purificada e caracterizada, comporta-se de modo idêntico à troponina I selvagem. / Muscle contraction results from the interaction of myosin with actin and this interaction is controlled by the calcium ion concentration which acts on the regulatory system associated with the actin filaments. This regulatory system comprises one tropomyosin molecule and a complex composed of three polypeptide subunits. One of this subunits, troponin C binds calcium ions; another, troponin T, binds to tropomyosin while the third, troponin I, inhibits the ATPase activity of actomyosin. This dissertation describes the development of the methodology to express troponin I in bacteria and its subsequent purification and characterization. Starting with a troponin I cDNA from chicken skeletal muscle, a pET expression vector was constructed. The initial vector resulted the expression of a troponin I fusion protein having an additional 18 amino acids. This protein was purified and characterized and found to behave like wild type troponin I. Subsequently, employing site-directed mutagenesis, a new vector was made which allowed the expression of the protein starting at its initial methionine codon and lacking the extra amino acids. However, no expression was achieved using this vector and, to circumvent this, further mutations were introduced in the first 25 codons which substituted AGG for CGT without altering the amino acid sequence. This final construct permitted the expression of a non fusion troponin I which, after purification and characterization, was found to behave identically to the wild type protein.

Da interação da actina com a miosina resulta a contração muscular. Esta interação é controlada pela concentração de íons cálcio, que atuam sobre um sistema regulatório associado ao filamento de actina. O sistema regulatório é composto de uma molécula de tropomiosina e um complexo protéico composto de três polipeptídeos chamado troponina. Uma das subunidades, troponina C, e o receptor de ions cálcio; outra, troponina T, liga-se à tropomiosina; e a terceira, troponina I, é a responsável pela inibição da atividade ATPásica da actomiosina. Esta dissertação descreve o desenvolvimento do processo de expressão da troponina I em bactéria, sua purificação e caracterização. Partindo de um cDNA de troponina I músculo esquelético de galinha, procedemos à construção de um vetor de expressão da proteína em E.coli com o sistema pET. Inicialmente construímos um vetor de expressão capaz de produzir a troponina I fundida a 18 aminoácidos. A proteína foi purificada e caracterizada, comportando-se de forma semelhante à troponina I selvagem. Numa segunda etapa, foram feitas mutações sítio-dirigidas para a construção de um novo sítio de restrição que possibilitou construir um vetor de expressão da troponina I a partir de sua metionina inicial. Entretanto não foi obtida expressão com esta construção. Para solucionar o problema, foram feitas novas mutações que alteraram os codons AGG presentes nos 25 primeiros codons do gene da proteína por codons CGT, sem alterar o aminoácido codificado. Nesta construção final foi obtida a expressão da proteína sem fusão, que purificada e caracterizada, comporta-se de modo idêntico à troponina I selvagem. / Muscle contraction results from the interaction of myosin with actin and this interaction is controlled by the calcium ion concentration which acts on the regulatory system associated with the actin filaments. This regulatory system comprises one tropomyosin molecule and a complex composed of three polypeptide subunits. One of this subunits, troponin C binds calcium ions; another, troponin T, binds to tropomyosin while the third, troponin I, inhibits the ATPase activity of actomyosin. This dissertation describes the development of the methodology to express troponin I in bacteria and its subsequent purification and characterization. Starting with a troponin I cDNA from chicken skeletal muscle, a pET expression vector was constructed. The initial vector resulted the expression of a troponin I fusion protein having an additional 18 amino acids. This protein was purified and characterized and found to behave like wild type troponin I. Subsequently, employing site-directed mutagenesis, a new vector was made which allowed the expression of the protein starting at its initial methionine codon and lacking the extra amino acids. However, no expression was achieved using this vector and, to circumvent this, further mutations were introduced in the first 25 codons which substituted AGG for CGT without altering the amino acid sequence. This final construct permitted the expression of a non fusion troponin I which, after purification and characterization, was found to behave identically to the wild type protein.

Dans les dernières années, une explosion de la recherche sur les ARN a eu lieue à cause de nombreuses découvertes démontrant l’importance de l’ARN dans plusieurs processus biologiques. Ainsi, de grandes quantités d’ARN sont devenues indispensables au bon déroulement de plusieurs études, notamment pour la biologie structurale et la caractérisation fonctionnelle. Cependant, il existe encore peu de méthodes de purification simples, efficaces, fiables et produisant un ARN sous forme native. Dans les dernières années, le laboratoire Legault a mis au point une méthode de purification par affinité utilisant une étiquette ARiBo pour la purification d’ARN transcrits in vitro par la polymérase à ARN du phage T7. Cette méthode de purification d’ARN a été spécifiquement développée pour maximiser la pureté et le rendement. De plus, elle est très rapide et fonctionne avec plusieurs types d’ARN. Cependant, comme plusieurs autres méthodes de purification, cette méthode produit des ARN avec des extrémités 5′ hétérogènes. Dans ce mémoire, des solutions sont proposées pour remédier au problème d’hétérogénéité en 5ʹ′ des ARN transcrits avec la polymérase à ARN du phage T7 et purifiés par la méthode ARiBo. La première solution consiste à choisir la séquence en 5′ parmi celles des 32 séquences testées qui ne présentent pas d’hétérogénéité en 5ʹ′. La seconde solution est d’utiliser une étiquette clivable en 5ʹ′ de l’ARN d’intérêt, tel que le ribozyme hammerhead, déjà utilisée pour ce genre d’application, ou le système CRISPR/Cse3 que nous proposons dans l’article présenté dans ce mémoire. De plus, nous avons adapté la méthode ARiBo pour rendre possible la purification d’un long ARN de 614 nt, le polycistron miR-106b-25. Nous avons également démontré la possibilité d’utiliser la méthode ARiBo pour l’isolation de protéines qui se lient à un ARN donné, le précurseur de miRNA pre-miR-153-2. En conclusion, ce mémoire démontre la possibilité d’adapter la méthode ARiBo à plusieurs applications. / In recent years, the field of RNA research has exploded due to several discoveries demonstrating the importance of RNA in many biological processes. Along with the increased interest in this field, large amounts of RNA have become essential to the success of several studies, in particular for structural biology and functional characterization. However, there are still very few native purification methods that are simple, efficient and reliable. In the past few years, the Legault laboratory has established an affinity purification method using an ARiBo tag to purify RNAs produced by in vitro transcription with the T7 RNA polymerase. This RNA purification method was specifically developed to maximise purity and yield. In addition, this method is fast and works with several types of RNAs. However, like several other purification methods, this method produces RNAs with 5' heterogeneity. This Master’s thesis propose solutions to overcome the problem of 5' heterogeneity for RNAs transcribed with the T7 RNA polymerase and purified with the ARiBo method. The first solution proposed is to choose a 5' sequence among those of the 32 sequences tested that do not present 5'- heterogeneity. The other possibility is the use of a cleavable tag at the 5'-end of the RNA of interest, such as the hammerhead ribozyme, already used for this purpose or the CRISPR/Cse3 system, which is presented here. Furthermore, we have adapted the ARiBo method to purify an RNA of 614 nt, the miRNAs cluster miR- 106b-25. We also demonstrate the possibility to use the ARiBo method to isolate proteins that bind a given RNA, the miRNA precursor pre-miR-153-2. In conclusion, this Master’s thesis demonstrates the possibility of adapting the ARiBo method for several applications.

The development of shape in multicellular organisms has intrigued human minds for millenia. Empowered by modern genetic techniques, molecular biologists are now striving to not only dissect developmental processes, but to exploit their modularity for the design of custom living systems used in bioproduction, remediation, and regenerative medicine. Currently, our capacity to harness this potential is fundamentally limited by a lack of spatiotemporal control over gene expression in multicellular systems. While several synthetic genetic circuits for control of multicellular patterning have been reported, hierarchical induction of gene expression domains has received little attention from synthetic biologists, despite its fundamental role in biological self-organization. In this thesis, I introduce the first synthetic genetic system implementing population-based AND logic for programmed hierarchical patterning of bacterial populations of Escherichia coli, and address fundamental prerequisites for implementation of an analogous genetic circuit into the emergent multicellular plant model Marchantia polymorpha. In both model systems, I explore the use of bacteriophage T7 RNA polymerase as a gene expression engine to control synthetic patterning across populations of cells. In E. coli, I developed a ratiometric assay of bacteriophage T7 RNA polymerase activity, which I used to systematically characterize different intact and split enzyme variants. I utilized the best-performing variant to build a three-color patterning system responsive to two different homoserine lactones. I validated the AND gate-like behavior of this system both in cell suspension and in surface culture. Then, I used the synthetic circuit in a membrane-based spatial assay to demonstrate programmed hierarchical patterning of gene expression across bacterial populations. To prepare the adaption of bacteriophage T7 RNA polymerase-driven synthetic patterning from the prokaryote E. coli to the eukaryote M. polymorpha, I developed a toolbox of genetic elements for spatial gene expression control in the liverwort: I analyzed codon usage across the transcriptome of M. polymorpha, and used insights gained to design codon-optimized fluorescent reporters successfully expressed from its nuclear and chloroplast genomes. For targeting of bacteriophage T7 RNA polymerase to these cellular compartments, I functionally validated nuclear localization signals and chloroplast transit peptides. For spatiotemporal control of bacteriophage T7 RNA polymerase in M. polymorpha, I characterized spatially restricted and inducible promoters. For facilitated posttranscriptional processing of target transcripts, I functionally validated viral enhancer sequences in M. polymorpha. Taking advantage of this genetic toolbox, I introduced inducible nuclear-targeted bacteriophage T7 RNA polymerase into M. polymorpha. I showed implementation of the bacteriophage T7 RNA polymerase/PT7 expression system accompanied by hypermethylation of its target nuclear transgene. My observations suggest operation of efficient epigenetic gene silencing in M. polymorpha, and guide future efforts in chassis engineering of this multicellular plant model. Furthermore, my results encourage utilization of spatiotemporally controlled bacteriophage T7 RNA polymerase as a targeted silencing system for functional genomic studies and morphogenetic engineering in the liverwort. Taken together, the work presented enhances our capacity for spatiotemporal gene expression control in bacterial populations and plants, facilitating future efforts in synthetic morphogenesis for applications in synthetic biology and metabolic engineering.