RNA catalysis is of fundamental importance in many biological functions, such as the peptidyl transferase activity of the ribosome and genetic control by riboswitches, among others. Small ribozymes are a convenient system to increase our understanding about the structure, folding and catalytic mechanism of ribozymes. This dissertation includes analysis of certain aspects of the catalytic mechanism in the hairpin and hammerhead ribozyme. In the hairpin ribozyme, we studied the functional consequences of molecular substitutions at two conserved positions, A9 and A10. These nucleotides are located close to the scissile phosphate but their exact function is unclear since they do not appear to be making any essential interactions with other nucleotides in the catalytic core. G, C, U, 2-aminopurine, 2, 6-diaminopurine, purine, and inosine were substituted at A9 and A10 and their effects on cleavage and ligation rates were analyzed. The effect of the variations on tertiary structure and docking was monitored by hydroxyl radical footprinting and native gel electrophoresis. It was observed that all the variants that exhibited poor docking and/or tertiary structure formation were also ligation challenged whereas they performed normally in the cleavage reaction. We found a unique variant, A10G that cleaved five times faster than A10 but did not exhibit any ligation. Results suggested that ligation required a more kinetically stable core than that needed to carry out cleavage. The hammerhead ribozyme field featured extensive disagreements between the crystal structure of the minimal hammerhead released in the mid 90s and the accumulating biochemical data. Much of the conflict was resolved with the new crystal structure of the extended hammerhead ribozyme. This structure confirmed many of the biochemical findings and brought out some new interactions, notably the G8·C3 base pair. We studied numerous base substitutions to establish the importance of the base pair for cleavage and ligation. Catalysis requires the formation of the base pair but even the fastest base paired variant was 10-fold slower than G8·C3 base pair. Docking and tertiary structure analysis by hydroxyl radical footprinting and native gel electrophoresis emphasized the importance of having a purine at position 8 and a pyrimidine at 3. Catalysis in the unmodified ribozyme was uniquely accompanied by hydrolysis of the 2′, 3′- cyclic phosphate ring present on one of the cleavage products, leading to the generation of non-ligatable products during a ligation assay. We determined the ligation rate-pH profile for unmodified ribozyme that differed from the cleavage rate-pH profile only at high pH.

AIDS is caused by a lentivirus, HIV-1. In addition to antiretroviral drugs that are currently in use for HIV/AIDS therapy, a number of gene therapy strategies have been designed as alternative therapies. Most of these therapies target HIV RNA/proteins, which are subject to high rate of mutation, resulting in escape mutants. Viral entry is mediated by CCR5 co-receptor in most routes of transmission. To downregulate CCR5 as a gene therapy approach, we targeted seven unique sites within the CCR5 mRNA by a multimeric hammerhead ribozyme, Rz1-7. Hammerhead ribozyme is a small RNA that cleaves a target RNA upon binding to it. Expressing the Rz1-7 from HIV-1- and MSCV-based vectors in otherwise susceptible cells inhibited replication of a CCR5-tropic strain of HIV-1 by 99-100%. The Rz1-7 will be tested for inhibition of HIV-1 replication in the CD4+ T-lymphoid and myeloid progeny of transduced human CD34+ hematopoietic progenitor stem cells. It may be preferable to interfere HIV-1 life cycle at the DNA level since a one-time inactivation might suffice to confer a complete and permanent inhibition of virus replication in the gene modified cells and their progeny. This is what other strategies that target the HIV-1 RNA/protein can hardly offer. For this purpose, group II introns, which are able to splice out and get incorporated into a specific DNA sequence, can be designed/modified to gain novel DNA targeting specificities. As a novel approach, we have examined whether insertion of a modified intron into an infectious HIV-1 clone at two sites within the integrase domain of HIV-1 pol gene could inhibit virus replication. Intron insertion into the HIV-1 clone was induced and mammalian cells were transfected with intron-inserted HIV-1 clones. Although similar amounts of HIV-1 RNA, protein, and progeny virus were produced from the clones as from wild-type HIV-1 provirus DNA, in the absence of a functional integrase, the HIV-1 reverse-transcribed DNA failed to integrate and virus replication was aborted. These results demonstrate that modified group II introns can confer complete inhibition of virus replication at the level of second round of infection. We are now developing vectors to assess whether intron insertion can take place in mammalian cells.

AIDS is caused by a lentivirus, HIV-1. In addition to antiretroviral drugs that are currently in use for HIV/AIDS therapy, a number of gene therapy strategies have been designed as alternative therapies. Most of these therapies target HIV RNA/proteins, which are subject to high rate of mutation, resulting in escape mutants. Viral entry is mediated by CCR5 co-receptor in most routes of transmission. To downregulate CCR5 as a gene therapy approach, we targeted seven unique sites within the CCR5 mRNA by a multimeric hammerhead ribozyme, Rz1-7. Hammerhead ribozyme is a small RNA that cleaves a target RNA upon binding to it. Expressing the Rz1-7 from HIV-1- and MSCV-based vectors in otherwise susceptible cells inhibited replication of a CCR5-tropic strain of HIV-1 by 99-100%. The Rz1-7 will be tested for inhibition of HIV-1 replication in the CD4+ T-lymphoid and myeloid progeny of transduced human CD34+ hematopoietic progenitor stem cells. It may be preferable to interfere HIV-1 life cycle at the DNA level since a one-time inactivation might suffice to confer a complete and permanent inhibition of virus replication in the gene modified cells and their progeny. This is what other strategies that target the HIV-1 RNA/protein can hardly offer. For this purpose, group II introns, which are able to splice out and get incorporated into a specific DNA sequence, can be designed/modified to gain novel DNA targeting specificities. As a novel approach, we have examined whether insertion of a modified intron into an infectious HIV-1 clone at two sites within the integrase domain of HIV-1 pol gene could inhibit virus replication. Intron insertion into the HIV-1 clone was induced and mammalian cells were transfected with intron-inserted HIV-1 clones. Although similar amounts of HIV-1 RNA, protein, and progeny virus were produced from the clones as from wild-type HIV-1 provirus DNA, in the absence of a functional integrase, the HIV-1 reverse-transcribed DNA failed to integrate and virus replication was aborted. These results demonstrate that modified group II introns can confer complete inhibition of virus replication at the level of second round of infection. We are now developing vectors to assess whether intron insertion can take place in mammalian cells.

Nucleic acids are important bio-macromolecules in living systems. They are involved in important functions like gene expression and regulation. Nucleoside triphosphates serve as precursors for biochemical synthesis of modified nucleic acids and nucleotide coenzymes. The modification of nucleic acids, particularly at nucleobases, can expand the function and chemical properties of nucleic acid. Herein, we report the chemical synthesis of a novel thiol-modified nucleoside S-(3-(acetylthio)propyl)-5-(mercaptomethyl)-uridine and the corresponding nucleotide via a “new synthetic methodology” developed in our laboratory. The synthesized triphosphate was used for RNA transcription. The activity and nuclease resistance of the transcribed RNA is studied. The results showed that the properties of the nucleotide with thiol functionality are as good as the native. The modified RNA can be used for RNA/protein complex structure studies and gold nanoparticles stabilizer. They can also serve as a probe in DNA/RNA microchip surface functionalization for detection of various diseases and pathogens.

The RNA World Hypothesis posits that RNA enzymes (ribozymes) catalyzed biochemical reactions in primitive cells prior to the emergence of proteins. However, the evolutionary mechanisms that gave rise to functional RNA sequences on early Earth remains largely unclear. Using a bottom-up approach that combines in vitro selection and high-throughput sequencing, we demonstrate how a self-cleaving RNA enzyme, the Hammerhead Ribozyme (HHR), may have evolved from non-catalytic sequences in the RNA World. Multiple starting libraries were generated by progressively increasing the number of randomized positions in the ribozyme’s catalytic core. The HHR was selected from each of these libraries following several rounds of amplification and enrichment. Deep sequencing analysis was then used to track evolutionary trends that gave rise to the wild-type sequence during each selection. This novel approach revealed a wide range of functional HHR variants. Notably, we discovered active hammerhead variants with mutations to previously identified essential nucleotides, shedding new light on the sequence requirements of the full-length, cis-acting ribozyme. We also demonstrate that the evolutionary trajectory of each nucleotide in the catalytic core directly correlates with their functional importance, potentially giving researchers a novel method to assess the sequence requirements of functional nucleic acids. Altogether, the in vitro evolution of ribozymes shows how complex molecules might have emerged from non-catalytic polymers in the RNA world, contributing to our understanding of the origin of life on Earth. / Thesis / Master of Science (MSc) / The origin of life is complicated by the interdependence between deoxyribonucleic acid (DNA), which stores genetic information, and protein, which performs essential cellular functions. The RNA World Hypothesis attempts to solve this paradox by underpinning ribonucleic acid (RNA) as the foundation of cellular based-life, due to its unique ability to store genetic information as well as perform complex chemical reactions. However, the way that functional RNA molecules (ribozymes) emerged on early Earth in the first place remains largely unclear. We simulated molecular evolution in the laboratory using a process known as in vitro selection to demonstrate how a self-cleaving RNA enzyme, the Hammerhead Ribozyme (HHR), may have evolved in the RNA World. We also discovered different versions of the HHR, shedding new light on its structure and function. Altogether, the results from this work pave the way for a deeper understanding of ribozyme evolution and the origins of life on Earth.

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.