1 |
Template Directed Ligation of RNA OligomersTurner, Eric January 2018 (has links)
The key to the RNA world hypothesis is the ribozyme, an information and catalytic agent that preceded proteins and DNA. Prior to ribozymes the sequences of RNA needed to build up to a length that could potentially be a ribozyme. This research focuses on computational modelling of hydrolysis, polymerization, and template-directed ligation to determine sequence patterns and characteristics that may have emerged due to these simple processes. A model containing L- and D-chirality monomers is used that incorporates the advantage of being a uniform chirality to achieve chiral symmetry breaking. Another chirality model is used where being uniform provides no advantage and a symmetry breaking still occurs. Beyond chirality we look at nucleobase models where we use a two letter alphabet containing adenine and uracil to determine symmetry breaking in sequence space. This results in self-complementary sequences dominating this model at all ligation rates but under certain initial conditions including high concentration, other types of sequences can be dominant. If a third base, guanine is added to this model a wobble base is created. In these models the self-complementary sequences containing uracil are the most prevalent due to uracil’s ability to pair with both adenine and guanine. Finally, upon adding a fourth base to the model guanine also becomes a wobble pair and the sequences containing uracil and guanine dominate the system for low ligation rates but at higher rates the uniform uracil and guanine sequences dominate. For each model a version is run with the templating reaction scaling linearly with the number of binding sites and without, where all templates are equally good. Generally, the scaling causes symmetry breakings at lower ligation values for each model. / Thesis / Master of Science (MSc) / The origin of life on Earth is a long-debated question that has been asked by nearly every civilization to have existed. This research addresses the origin of life in the context of the RNA World theory, which proposes that the first kind of replicating molecules were RNA strands, specifically, catalytic RNA sequences, called ribozymes. We carry out computer simulations of the formation and break-up of short RNA strands. Strands can grow by joining together randomly, or due to the action of template strands. We find that, if this process occurs repeatedly, the RNA strands in the mixture move towards states in which groups of sequences that are good templates for one another occur together at high concentrations. By studying the possible states that arise in this reaction mixture, we hope to learn about the first replicating RNA strands that lead to the origin of life.
|
2 |
Computational Modeling of RNA Replication in an RNA WorldTupper, Andrew January 2020 (has links)
The biology of modern life predicts the existence of an ancient RNA world. A phase of evolution in which organisms utilized RNA as a genetic material and a catalyst. However, the existence of an RNA organism necessitates RNA’s ability to self-replicate, which has yet to be proven. In this thesis, we utilize computational modeling to address some of the problems facing RNA replication. In chapter 2, we consider a polymerase ribozyme replicating by the Qβ bacteriophage mechanism. When bound to a surface, limited diffusion allows for survival so long as the termination error rate is below an error threshold. In Chapter 3, we consider the replication of short oligomers through an abiotic mechanism proposed in prebiotic experiments. When limited by substrate availability, competition results in the emergence of uniform RNA polymers from a messy prebiotic soup containing nucleotides of different chirality and sugars. In chapter 4, we consider the possibility of an RNA world lacking cytosine. Without cytosine, the ability of RNA to fold to complex secondary structures is limited. Furthermore, G-U wobble base pairing hinders the transfer of information during replication. Nevertheless, we conclude that an RNA world lacking cytosine may be possible, but more difficult for the initial emergence of life. In chapter 5, we analyze abiotic and viral mechanisms of RNA replication using known kinetic and thermodynamic data. While most mechanisms fail under non-enzymatic conditions, rolling-circle replication appears possible. In chapter 6, we extend our analysis of the rolling-circle mechanism to consider the fidelity of replication. Due to the thermodynamic penalty of incorporating an error, rolling-circle replication appears to undergo error correction. This results in highly accurate replication and circumvents Eigen’s paradox. Rolling-circle replication therefore presents an appealing option for the emergence of RNA replication in an RNA world. / Thesis / Doctor of Philosophy (PhD)
|
3 |
Evolving an Enzyme From a Non-Catalytic SequenceGysbers, Rachel January 2015 (has links)
Life would not exist in the absence of catalysis. The “RNA World” model for the origin of life hinges on the capabilities of ribonucleic acid to encode information and perform catalysis (i.e. self-replication). Previously, functional nucleic acids such as ribozymes and deoxyribozymes (DNAzymes) have been isolated using the process of in vitro selection. This method is typically performed by isolating a catalytically active molecule from a large random library, with the assumption being that active molecules are already present in the pool and this method filters them from inactive molecules. However, in vitro selection has never been used to show that a molecule can be evolved from an inactive to an active catalyst. Here we show that the properties of DNA can be exploited to act as a proxy system for the origins of biotic chemistry by isolating a functional catalyst from a previously non-catalytic sequence. This project employs a novel perspective; rather than a random library, a known, non-functional sequence is utilized. Using in vitro selection, this known sequence is gradually evolved into a functional catalyst by solely allowing the existence of sequences that acquire mutations which enhance their function. Deep sequencing analysis of DNA pools along the evolution trajectory has identified individual mutations as the progressive drivers of molecular evolution. Evolving a catalyst from a non-catalyst gives insight into the comprehension of how life originated. This project demonstrates that an enzyme can indeed arise from a sequence of a functional polymer via permissive molecular evolution, a mechanism that may have been exploited by nature for the creation of the enormous repertoire of enzymes in the biological world today. / Thesis / Master of Science (MSc)
|
4 |
Simulations of Non-Enzymatic Template Directed RNA ReplicationChamanian, Pouyan January 2022 (has links)
The universal traits of cellular expression and replication in modern life point to the existence of an ancient RNA world. Leading up to the origin of life, this stage of evolution utilized RNA as the genetic material, and as a catalyst in the form of ribozymes. Although it is expected that a polymerase ribozyme was required for the efficient replication of RNA, it is also likely that the earliest form of replication took place under non-enzymatic conditions. There are several problems with the current scenarios depicting non-enzymatic RNA replication, thus we aim to examine them in more detail using computational models. We first consider the relationship between the thermodynamics of RNA base pairing and non-enzymatic nucleotide addition in an attempt to model the rate of primer extension. Our predicted rates reveal the model parameters to be too simple to produce reliably accurate results. For now, we should simply use available experimental rate data, until we have access to more data and less unknown parameters. Nevertheless, the model indicates that the primer extension rate does depend on thermodynamics of base pairing, and a more accurate model can be of great use when creating realistic complex models of RNA world scenarios. In chapter 3, we investigate non-enzymatic RNA replication under temperature cycling using computer simulations. When starting with a diverse mixture of sequences, partially matching sequences can reanneal in configurations that allow continued strand growth. This is in contrast to the case of having multiple copies of matching sequences, where reannealing occurs quickly upon cooling. We find that, starting with short oligomers, strands can grow over multiple cycles to produce long sequences over 100 nucleotides in length. The small strand extension per cycle does not produce replicates of any one specific sequence. This relates to the work done in chapter 4, where we look for the presence of a virtual circular genome within our simulations. In a virtual circle, short overlapping RNA sequences will make up a mutually catalytic set. Within the diversity of our simulation, virtual circles are rare, and require a specific level of starting mixture diversity along with no input of new sequences. Continued replication of the diverse sequence mixture and emergence of long strands may eventually lead to the creation of rolling circles and ribozymes. / Thesis / Master of Science (MSc) / The origin of biological life can be traced back by looking at the common themes between modern cellular processes. The role of RNA polymers seems to be of great importance, making us believe that an RNA world existed leading up to life’s origin. During this time, RNA would act as both a genetic material and a catalyst. To examine this theory in more detail, we use computational modeling to recreate and explore the various potential chemistries and conditions on the early Earth. Specifically, we explore the problems that exist for the replication and production of RNA polymers. Our results can be used to guide future theoretical and experimental research of the RNA world.
|
5 |
Prebiotic Synthesis of Pyrimidine NucleosidesCollins, James P. 28 November 2005 (has links)
The problem of forming a glycosidic bond between ribose and the free nucleoside bases to produce beta-nucleosides under plausible prebiotic conditions is commonly referred to in origin of life research as The Nucleoside Problem. The lack of a general solution to this problem currently represents one of the largest stumbling blocks to the RNA world hypothesis and many other theories regarding the origin of life. Over thirty years ago the purine nucleosides were successfully synthesized by drying the fully-formed bases and ribose together in the presence of divalent metal ion salts. However, glycosidic bond formation by the pyrimidine bases has never been achieved under similar reaction conditions. This thesis describes the first plausible prebiotic synthesis of a pyrimidine nucleoside, demonstrated with the pyrimidine base analogue 2-pyrimidinone. Information provided by nucleoside-formation reaction involving 2-pyrimidinone and related pyrimidine bases should provide valuable insights into the possible mechanism by which glycosidic bond formation was accomplished on the prebiotic Earth.
|
6 |
Characterization of Supramolecular Polymer Systems Composed of Prebiotically Plausible Recognition UnitsKhanam, Jaheda 08 August 2014 (has links)
Supramolecular polymers have a practical impact on the healthcare field as they can act as scaffolds to repair parts of organs such as the brain or heart. In addition, they can provide insight into theories relating to the origins of life. For instance, the hypothesis that RNA played a more important role in early biology, the RNA World hypothesis, would be strengthened if there were a way to show the spontaneous formation of RNA-like polymers from monomer units. However, the natural nucleobases do not assemble at the monomer level, nor do they form nucleosides readily with ribose, leading some to speculate that the first nucleobases may have been different from the ones used in biology today. This conundrum encouraged us to begin looking for alternative nucleobases that are able to self-assemble into polymers capable of storing information. Our lab has recently demonstrated that a modified 2,4,6-triaminopyrimidine (TAP) will assemble with cyanuric acid (CA) in water through interactions that are analogous to those between complementary nucleobases found in DNA and RNA. When TAP is modified at one of its three faces, it can pair through specific hydrogen bonding with CA on two of its faces, forming rosette structures. These rosettes self-assemble to form extremely long structures through the stacking of tens of thousands of rosettes. In this study we are investigating prebiotically relevant syntheses of TAP nucleosides. Using chromatography techniques and nuclear magnetic resonance we found that the unmodified TAP with D-ribose formed nucleosides in 60% yields with the major product (20%) being a C-nucleoside 5-β-ribofuranosyl-2,4,6-triaminoprymidine or TARC. TARC forms hydrogels with CA, both in the crude reaction and after purification, indicative of the formation of supramolecular polymers out of a complex mixture. The results of this study provide support for the possibility of pre-RNA molecules.
|
7 |
Ice as a medium for RNA-catalysed RNA synthesis and evolutionAttwater, James January 2011 (has links)
A critical event in the origin of life is thought to have been the emergence of a molecule capable of self-replication and evolution. According to the RNA World hypothesis, this could have been an RNA polymerase ribozyme capable of generating copies of itself from simple nucleotide precursors. In vitro evolution experiments have provided modern examples of such ribozymes, such as the R18 RNA polymerase ribozyme, exhibiting basic levels of this crucial catalytic activity; R18’s activity, however, falls far short of that required of an RNA replicase, leaving unanswered the question of whether RNA can catalyse its self-replication. This thesis describes the development and use of a novel in vitro selection system, Compartmentalised Bead-Tagging (CBT), to isolate variants of the R18 ribozyme with improved sequence generality and extension capabilities. CBT evolution and engineering of polymerase ribozymes, together with RNA template evolution, allowed the synthesis of RNA molecules over 100 nucleotides long, as well as the RNA-catalysed transcription of a catalytic hammerhead ribozyme. This demonstrates the catalytic capabilities of ribozyme polymerases. The R18 ribozyme was also exploited as an analogue of a primordial replicase, to determine replicase behaviour in different reaction environments. Substantial ribozyme polymerisation occurred at −7˚C in the liquid eutectic phase of water-ice; increased ribozyme stability at these low temperatures allowed longer extension products to be generated than at ambient temperatures. The concentration effect of eutectic phase formation could also yield RNA synthesis from dilute solutions of substrates, and provide quasicellular compartmentalisation of ribozymes. These beneficial physicochemical features of ice make it a potential protocellular medium for the emergence of primordial replicases. Ice also could serve as a medium for CBT, allowing the isolation of a polymerase ribozyme adapted to the low temperatures in the ice phase, demonstrating the primordial potential and modern feasibility of ribozyme evolution in ice.
|
8 |
Pre-evolutionary dynamics in autocatalytic RNA networks / Dynamique pré-évolutive des réseaux ARN autocatalytiquesArsene, Simon 12 October 2018 (has links)
Les réseaux de molécules interdépendantes sont depuis quelque temps considérés comme de potentiels candidats pour avoir amorcé la transition de la biologie à la chimie. Bien qu'ils aient été intensivement examinés en théorie, il n'existe toujours aucune preuve expérimentale pour confirmer ou infirmer leur supposé rôle crucial dans les origines de la vie. En particulier, il nous manque encore une démonstration empirique des trois ingrédients habituellement présentés comme requis pour l'évolution darwinienne: l'hérédité, la variation et la sélection. Un système qui posséderait les trois tout en étant couplé à un processus de réplication en compartiments serait théoriquement capable d’évoluer au sens darwinien du terme. Par exemple, cela a été montré théoriquement pour les Ensembles Collectivement Autocatalytiques (CAS pour Collectively Aucatalytic Sets en anglais) où chaque molécule de l'ensemble est formée catalytiquement par un autre membre de l'ensemble. Ici, nous utilisons le système de ribozyme Azoarcus, qui catalysent des réactions de recombinaisons, pour former expérimentalement des CASs structurellement divers afin d’explorer leurs propriétés évolutives. Dans ce système, les ribozymes peuvent catalyser la formation d'autres ribozymes à partir de fragments plus petits, présents dans l'environnement. Nous utilisons un dispositif de microfluidique en gouttes associé au séquençage haut-débit pour mener une étude à grande échelle sur des milliers de CASs Azoarcus. Nous développons une approche perturbative pour identifier les paramètres topologiques importants contrôlant les variations observées dans les CAS à la suite de perturbations de l’environnement, ici l'ajout d'une nouvelle espèce. Nous déterminons ensuite l’ensemble restreint de caractéristiques du réseau régissant la mémoire des conditions initiales dans les CASs Azoarcus, un prérequis pour l'hérédité, en utilisant un modèle théorique validé par des données expérimentales. Enfin, nous démontrons qu’il existe dans les CASs Azoarcus des processus cataboliques qui les rendent robustes aux perturbations des fragments qui composent leur substrat et donc plus pertinent d’un point de vue prébiotique. Ces résultats démontrent le rôle crucial des CASs à base d’ARN dans les origines de la vie et illustrent comment la structure de leur réseau peut être adaptée pour obtenir des CASs avec des propriétés intéressantes d’un point de vue évolutif, ouvrant la voie à une démonstration expérimentale de l'évolution darwinienne avec système purement moléculaire. / Networks of interdependent molecules are considered plausible candidates for initiating the transition from biology to chemistry. Though they have been intensively scrutinized theoretically, there is still no experimental evidence for confirming or denying their supposed crucial role in the origins of life. In particular, we are still lacking experimental proofs of any of the three ingredients usually presented as required for Darwinian evolution: heredity, variation and selection. A system that would possess the three while being coupled to some sort of encapsulated replication process would theoretically be able to undergo Darwinian evolution. As a matter of fact, this has been shown theoretically for Collectively Autocatalytic Sets (CAS) where each molecule of the set is catalytically formed by another member of the ensemble. Here we use the Azoarcus recombination ribozyme system to experimentally form structurally diverse CASs to explore their evolutionary properties. In this system, the ribozymes can catalyze the assembly of other ribozymes from smaller fragments, present in the food set. We first use a droplet microfluidics set-up coupled with next-generation sequencing to conduct a large scale study on thousands of Azoarcus CASs. We develop a perturbative approach to identify the important topological parameters that control variations in CASs as a result of environmental perturbations, here the addition of a new species. We then determine the small set of network features governing memory of the initial conditions in Azoarcus CAS, a pre-requisite for heredity, by using a computational model validated by experimental data. Finally, we demonstrate that Azoarcus CAS possess catabolic processes which make them robust to perturbations in the food set and thus more prebiotic relevant. These results provide evidence for the crucial role of RNA CASs in the origins of life and illustrate how the network structure can be tailored to obtain CASs with properties interesting from an evolutionary point of view, paving the way to an experimental demonstration of Darwinian evolution with a purely molecular system.
|
9 |
Comparing Protocell and Surface-Based Models of RNA Replicator Systems and Determining Favourable Conditions for Linkage of Functional Strands / Simulations of RNA Replicator SystemsShah, Vismay January 2019 (has links)
In hypothesized RNA-World scenarios, replication of RNA strands is catalyzed by error-prone polymerase ribozymes. Incorrect replication leads to the creation of non-functional, parasitic strands which can invade systems of replicators and lead to their death. Studies have shown two solutions to this problem: spatial clustering of polymerases in models featuring elements to limit diffusion, and group selection in models featuring protocells. Making a quantitative comparison of the methods using results from the literature has proven difficult due to differences in model design. Here we develop computational models of replication of a system of polymerases, polymerase complements and parasites in both spatial models and protocell models with near identical dynamics to make meaningful comparison viable. We compare the models in terms of the maximum mutation rate survivable by the system (the error threshold) as well as the minimum replication rate constant required. We find that protocell models are capable of sustaining much higher maximum mutation rates, and survive under much lower minimum replication rates than equivalent surface models. We then consider cases where parasites are favoured in replication, and show that the advantage of protocell models is increased. Given that a system of RNA strands undergoing catalytic replication by a polymerase is fairly survivable in protocell models, we attempt to determine whether isolated strands can develop into genomes. We extend our protocell model to include additional functional strands varying in length (and thus replication rate) and allow for the linkage of strands to form proto-chromosomes. We determine that linkage is possible over a broad range of lengths, and is stable when considering the joining of short functional strands to the polymerase (and the same for the complementary sequences). Moreover, linkage of short functional strands to the polymerase assures more cells remain viable post division by ensuing a good quantity of polymerase equivalents are present in the parent cell prior to splitting. / Thesis / Master of Science (MSc) / Collections of RNA polymers are good candidates for the origin of life. RNA is able to store genetic information and act as polymerase ribozymes allowing RNA to replicate RNA. Polymerases have been experimentally developed in labs, however none are sufficiently general to work well in an origins of life setting. These polymerases are vulnerable to mistakes during copying, making survival of RNA systems difficult. Such systems have been studied by computer simulations, showing that the strands need to be kept together for survival, either on surfaces or in primitive cells. Differences in the details of the models has made comparing the surfaces to cells difficult. This work creates a unified model base allowing for comparison of these two environments. We find that the existence of primitive cells is very beneficial to systems of RNA polymers and thus it is likely such cells existed at the origin of life.
|
10 |
Exploring Ligand Structure and Thermodynamics of the Malachite Green RNA AptamerDa Costa, Jason Bernard January 2012 (has links)
RNA aptamers are in vitro sequences of RNA that have a high affinity for their target ligand. They have applications in therapeutics, biosensors and molecular machines. While the practical applications of aptamers are increasing, it is important to study their structure and thermodynamics to improve the understanding of these molecular tools. The malachite green aptamer (MGA) provides a model system to study the interactions between aptamer and ligand that do not involve hydrogen bonding between ligand and receptor. While the original application of this aptamer was abandoned, study of the MGA binding pocket revealed an electronegative environment that was harnessed for catalysis. MGA binding also supported the notion that aptamers bind by adaptive binding. Adaptive binding is the ability of molecules to mold themselves around the structure of a ligand thereby incorporating it into their three-dimensional fold.
To further expand our understanding of MGA binding and to clarify conflicting reports of affinities, we conducted isothermal calorimetry binding studies. The results reveal that the entropy of complex formation plays a large role in determining binding affinity and ligand specificity. This data combined with previous structural studies show that metal ions are required to stabilize the complexes with non-native ligands, whereas, the complex with the original selection target is stable at low salt and in the absence of divalent metal ions. Next, competitive binding studies using isothermal titration calorimetry were conducted with the aim of understanding the adaptive nature of RNA. The results of these studies reveal that there are limits to the adaptability of the aptamer. Binding of one type of ligand reduces the affinity of the aptamer pocket to a differently shaped ligand, even if this second ligand has a significantly higher affinity.
The ability of MGA to change ligand preference based on buffer conditions, and the previously reported catalysis suggested that RNA may have a potential supporting multiple functions in the same molecule. To investigate this possibility we attempted to select an aptamer that supports both ligand binding and catalysis. By conducting both a DNA and RNA selection we hoped to add to the
iv
collection of DNA and RNA aptamers selected for the same target. There are currently too few of these to determine if any correlation can be made between DNA and RNA sequences that bind the same target. The target of the selection was fluorescein diacetate (FDA), which was chosen with the aim that it would allow the exploration of the inherent potential of the selected aptamer to cleave FDA to fluorescein. The RNA selection proved to be more successful and an attempt was made to characterize the binding of the aptamer to its target fluorescein diacetate. Unfortunately there were complications with the binding assays, but future work is proposed that should address the issues.
In order to expand the MGA catalytic repertoire attempts were made to synthesize new ligands that could exploit the catalytic potential of the MGA binding pocket. Unfortunately these attempts were unsuccessful, however further attempts are recommended. The MGA used in this study was transcribed in vitro using T7 RNA polymerase. This process is known to add extra nucleotides to the end of the transcription product. Attempts were made to eliminate the n+1 product by introducing a ribozyme or DNAzyme. These were met with difficulties resulting in low yield, however mass spectrometry revealed that n and n+1 MGA bind to ligand. This, along with secondary structure prediction suggests that MGA n+1 behaves the same as n.
Overall, the results presented here provide insights into the capabilities of RNA aptamers with respect to ligand binding and catalysis.
|
Page generated in 0.1078 seconds