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Error-Tolerant Coding and the Genetic CodeGutfraind, Alexander January 2006 (has links)
The following thesis is a project in mathematical biology building upon the so-called "error minimization hypothesis" of the genetic code. After introducing the biological context of this hypothesis, I proceed to develop some relevant information-theoretic ideas, with the overall goal of studying the structure of the genetic code. I then apply the newfound understanding to an important question in the debate about the origin of life, namely, the question of the temperatures in which the genetic code, and life in general, underwent their early evolution. <br /><br /> The main advance in this thesis is a set of methods for calculating the primordial evolutionary pressures that shaped the genetic code. These pressures are due to genetic errors, and hence the statistical properties of the errors and of the genome are imprinted in the statistical properties of the code. Thus, by studying the code it is possible to reconstruct, to some extent, the primordial error rates and the composition of the primordial genome. In this way, I find evidence that the fixation of the genetic code occurred in organisms which were not thermophiles.
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Error-Tolerant Coding and the Genetic CodeGutfraind, Alexander January 2006 (has links)
The following thesis is a project in mathematical biology building upon the so-called "error minimization hypothesis" of the genetic code. After introducing the biological context of this hypothesis, I proceed to develop some relevant information-theoretic ideas, with the overall goal of studying the structure of the genetic code. I then apply the newfound understanding to an important question in the debate about the origin of life, namely, the question of the temperatures in which the genetic code, and life in general, underwent their early evolution. <br /><br /> The main advance in this thesis is a set of methods for calculating the primordial evolutionary pressures that shaped the genetic code. These pressures are due to genetic errors, and hence the statistical properties of the errors and of the genome are imprinted in the statistical properties of the code. Thus, by studying the code it is possible to reconstruct, to some extent, the primordial error rates and the composition of the primordial genome. In this way, I find evidence that the fixation of the genetic code occurred in organisms which were not thermophiles.
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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
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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.
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Potential prebiotic roles of (amino-)acylation in the synthesis and function of RNAChan, Christopher K. W. January 2013 (has links)
The Sutherland group recently demonstrated that from a mixture of oligoribonucleotide-2'- or 3'-phosphates the latter is chemoselectively acetylated. This is shown to mediate a template-directed ligation to give predominantly 3',5'-linked RNA that is acetylated at the ligation junction (acetyl-RNA). It was suggested that RNA emerged prebiotically via acetyl-RNA and also is proposed to have favourable genotypic properties due to greater propensity to form duplex structure. To study the properties of acetyl-RNA, their synthesis by solid-phase chemistry was required and described is the design of a 2'/3'-O-acetyl orthogonal protecting group strategy. Key to the orthogonal protecting group strategy is the use of (2-cyanoethoxy)carbonyl for the protection of the nucleobase exocyclic amines and a photolabile solid-phase linker group that allowed partial on-column deprotection. The synthesis of the 2'/3'-O-acetyl and 2'/3'-O-TBDMS phosphoramidites, in addition to preparation of a photolabile solid-phase support, are described. With the materials to hand the procedures for an automated synthesis of acetyl-RNA were optimised and several acetyl-RNA oligonucleotides were synthesised. The duplex stability of acetyl-RNA with up to four sites of 2'-O-acetylation were assessed by UV melting curve analysis. Remarkably, the acetyl groups caused a consistent decrease in Tm of between 3.0-3.2 °C. Thermodynamic parameters indicated a decrease in duplex stability that was consistent with a decrease in hydration of the minor groove resulting in a reduction of the stabilising hydrogen bonding network. The stability of a tetraloop was also found to decrease on acetylation. The acetylated- tetraloop it is able to form duplex at lower concentrations than the natural tetraloop. Additionally, it is more stable at high concentrations, indicating that acetyl-RNA favours duplex over other secondary structure. These properties are considered to give acetyl-RNA competitive advantage for their non-enzymatic replication. Aminoacylation of RNA is an important process in modern biology but the intermediacy of aminoacyl-adenylates is considered to be prebiotically implausible. A potentially prebiotic aminoacylation of nucleoside-3'-phosphates, selective for the 2'-hydroxyl, is presented. However, it was thought the aminoacylation yields could be improved and so a search for an alternative activator was conducted. Oligoribonucleotide-3'-phosphates were exposed to the aminoacylation conditions and selective aminoacylation at only the 2'-hydroxyl of the 3'-end was observed. In particular, the aminoacylation of a trimer lends support to Sutherland’s theory of a linked origin of RNA and coded peptide synthesis.
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Studies towards the chemical origins of lifeIslam, Saidul January 2011 (has links)
The 'RNA World' hypothesis states that RNA was the first living system on the primitive Earth, where it carried out dual genotypic and phenotypic functions. Therefore, RNA must have self-assembled by purely chemical means from small prebiotic feedstock molecules. A plausible demonstration of the synthesis of RNA with the natural [5'→3'] phosphodiester linkage, and its self-replication has not been achieved so far. Some have speculated a 'simpler' informational polymer preceded it, and biology based on this polymer subsequently 'invented' RNA. The structurally simpler L-α-threofuranosyl nucleic acid (TNA) has been proposed as a primordial ancestor to RNA. A study into the potential self-assembly of TNA nucleotides was carried out. It is shown that as a direct result of TNA's structural simplicity, its generational chemistry is more difficult than RNA. The tetrose aminooxazolines are unstable under the conditions of its formation. The tetrose anhydronucleosides efficiently incorporate phosphate to form activated tetrose cytidine-2',3'-cyclic phosphates, but with the wrong stereochemistry. Strong support for the 'RNA world' hypothesis came from a report in 2009 of the prebiotic synthesis of activated pyridimine ribonucleoside-2',3'-cyclic phosphates. Oligomerisation studies were carried out on these activated monomers with various catalysts, and NMR studies were carried out to determine the aspects of their reactivity. It was found that only short oligomers are formed. However, nucleoside-2',3'-cyclic phosphates were found to selectively hydrolyse to a 2:1 mixture of 3' and 2'-monophosphates, and this observation was considered as etiologically relevant. Nucleoside-2' and 3'-monophosphates cyclise back to nucleoside-2',3'-cyclic phosphates upon phosphate activation, and so cannot be considered as direct candidates for oligomerisation. A chemistry that selectively uses the nucleoside-3'-phosphate for the synthesis of RNA, and recycles the unwanted 2'-phosphate would be highly desirable. Thus, a regio- and chemoselective reaction that selectively acetylates monomer and oligomer nucleoside-3'-phosphates at the 2'-hydroxyl in water is presented. Nucleoside-2'-phosphates are shown to acetylate less efficiently, and show a greater propensity to recyclise back to nucleoside-2',3'-cyclic phosphates. Purine nucleotides were also found to acetylate better than pyrimidines. This potentially primordial protecting group chemistry approach towards the prebiotic synthesis of RNA is conceptually novel, and has the potential to give a natural [5'→3'] phosphodiester linkage isomer. It is considered as a major step towards solving the long-standing problem of non-enzymatic self-replication of RNA.
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Elektrochemická analýza RNA: Vývoj metódy vhodnej pre charakterizáciu produktov neenzymatickej polymerácie cyklických nukleosid monofosfátov za podmienok modelujúcich prebiotické prostredie / Electrochemical analysis of RNA: development of a method suitable for the characterization of products of non-enzymatic polymerization of cyclic nucleoside monophosphates under conditions modeling prHesko, Ondrej January 2019 (has links)
This thesis focuses on the optimazation of the electrochemical method, which characterizes products of untemplated nonenzymatic polymerization of 3',5' -cyclic guanosine monophosphate (cGMP) under conditions modeling prebiotic environment. An adsorptive transfer stripping techniques on carbon electrode and gel electrophoresis were used. The method was optimized on the model system of oligonucleotides located in solution of cGMP on carbon electrode, where DNA and RNA adsorb. This technique allows simple removing of interfering substances such as cGMP, which are not present in the original sample, but they do not adsorb on the surface of electrode or they adsorb weaker than oligonucleotides or polynucleotides. Analyses are based on the selective desorption of cGMP from the surface of the carbon electrode by the chemical and physical methods before the measurement of linear voltammetry itself. Detergents, such as SDS, Tween 20 and Triton x-100 with different concentrations and electrostatic repulsions of cGMP with different negative potentials on the carbon electrode were used for the selective desorption of cGMP. The selective desorption of cGMP was observed for all detergents and inserted negative potentials. Used methods were compared and the most effective detergent for selective desorption of cGMP was SDS. Desorption of oligonucleotides was minimalized by inserted positive potential on washed carbon electrode in 0,01% SDS in basic medium. This optimized method was used on electrochemical analysis of preliminary samples of untemplated nonenzymatic polymerization of 3',5' -cGMP and compared to the analysis of gel electrophoresis.
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Synthèse d'ARN en conditions prébiotiques / RNA synthesis in prebiotic conditionsDa Silva, Laura 01 December 2016 (has links)
L'une des questions essentielles concernant l'origine de la vie est de comprendre les étapes de l'évolution ayant permis le passage d’une chimie prébiotique complexe aux premières étapes biologiques. Les constituants cellulaires actuels nous permettent de suivre le lien des produits chimiques aux métabolites biochimiques, de l'ancien au monde moderne. De nombreuses preuves soutiennent l'hypothèse du "monde à ARN" stipulant qu’au début de l'évolution de la vie, l'ARN était responsable à la fois du stockage et du transfert de l'information génétique mais aussi de la catalyse de réactions biochimiques. Dans cette thèse, j’ai étudié la synthèse non-enzymatique d’ARN dans des conditions prébiotiques. Ces conditions simulent les processus hydrothermaux qui se produisent dans les sources chaudes hydrothermales modernes et étaient vraisemblablement omniprésentes sur la Terre primitive. J’ai ensuite recherché les conditions optimales permettant la synthèse de longs polymères d’ARN et montré que la présence d’une matrice cristalline augmente de manière significative le rendement et la longueur des polymères synthétisés. Enfin, j’ai examiné la stabilité des polymères nouvellement synthétisés dans ces conditions hydrothermales and comparé la dégradation par dépurination des nucléotides et polymères en présence des deux agents organisateurs favorisant la polymérisation, soit des phospholipides, soit des sels cristallins. Nous avons conclu que, bien que la décomposition des nucléotides ait lieu dans ces conditions, des nucléotides puriques sont toujours disponibles pour participer à la synthèse de polymères et que la présence de phospholipides protège les nucléotides contre la dégradation. / One remaining crucial point in the early life history is to understand how evolution passed from complex prebiotic chemistry to simple biology. Current cellular facts allow us to follow the link from chemical to biochemical metabolites, from the ancient to the modern world. A substantial weight of evidence supports the “RNA world” hypothesis stipulating that the earliest forms of life passed through a phase in which RNA served both for the storage and transfer of genetic information and for the catalysis of biochemical reactions. In this thesis, we studied the non-enzymatic synthesis of RNA under simulated prebiotic conditions. These conditions simulated hydrothermal processes that commonly occur in volcanic hydrothermal fields today and were presumably ubiquitous on the primitive Earth. We then looked for optimal conditions allowing synthesis of long RNA-like polymers and showed that the presence of crystalline matrix significantly increases the yield and length of synthesized polymers. Finally, we investigated the stability of the synthesized polymers under hydrothermal conditions and compared the degradation by depurination of nucleotides and polymers in the presence of two organsing agents that promotes polymerisation, either phospholipids or salts crystals. We concluded that although decomposition of nucleotides occurs in these simulated conditions, purine nucleotides are still available to participate in polymers synthesis and the presence of phospholipids protects nucleotides against degradation.
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The Origin of Life by Means of Autocatalytic Sets of BiopolymersWu, Meng 10 1900 (has links)
<p>A key problem in the origin of life is to understand how an autocatalytic, self-replicating biopolymer system may have originated from a non-living chemical system. This thesis presents mathematical and computational models that address this issue. We consider a reaction system in which monomers (nucleotides) and polymers (RNAs) can be formed by chemical reactions at a slow spontaneous rate, and can also be formed at a high rate by catalysis, if polymer catalysts (ribozymes) are present. The system has two steady states: a ‘dead’ state with a low concentration of ribozymes and a ‘living’ state with a high concentration of ribozymes. Using stochastic simulations, we show that if a small number of ribozymes is formed spontaneously, this can drive the system from the dead to the living state. In the well mixed limit, this transition occurs most easily in volumes of intermediate size. In a spatially-extended two-dimensional system with finite diffusion rate, there is an optimal diffusion rate at which the transition to life is very much faster than in the well-mixed case. We therefore argue that the origin of life is a spatially localized stochastic transition. Once life has arisen in one place by a rare stochastic event, the living state spreads deterministically through the rest of the system. We show that similar autocatalytic states can be controlled by nucleotide synthases as well as by polymerase ribozymes, and that the same mechanism can also work with recombinases, if the recombination reaction is not perfectly reversible. Chirality is introduced into the polymerization model by considering simultaneous synthesis and polymerization of left- and right-handed monomers. We show that there is a racemic non-living state and two chiral living states. In this model, the origin of life and the origin of homochirality may occur simultaneously due to the same stochastic transition.</p> / Doctor of Philosophy (PhD)
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The Emergence of the RNA World on the Early EarthPearce, Ben K. D. January 2017 (has links)
Life on Earth likely began as an RNA world, where cell-free or compartmentalized ribonucleic acid (RNA) molecules dominated as the replicating and evolving lifeforms prior to the emergence of DNA- and protein-based life. The focus of this thesis is on when and how this RNA world emerged. We use astrophysical and geophysical studies to constrain when the Earth was habitable, and biosignature studies to constrain when the Earth was inhabited. From this we obtain a time interval for the emergence of life. Considering all these constraints, we find that the Earth was habitable as early as 4.5 Ga, or as late as 3.9 Ga, depending on whether the early influx of asteroids inhibited life from emerging. The time that the Earth was inhabited is more precisely constrained to 3.7 Ga. This suggests life emerged within 800 Myr, and possibly in < 200 Myr. Between 4.5–3.7 Ga, the continental crust was slowly rising up from the global ocean, providing dry land on which warm little ponds could form. We develop the theory for the emergence of RNA polymers in these pond environments, whose wet-dry cycles promote polymerization. RNA is comprised of chains of nucleotides, and the latter is made up of ribose, phosphate, and a characteristic nucleobase. We numerically model the survival and evolution of nucleobases in warm little ponds from meteorite and interplanetary dust sources. The wet-dry cycles of our ponds are controlled by precipitation, evaporation, and seepage. The nucleobase sinks include photodissociation, seepage, and hydrolysis. Nucleobase and nucleotide seepage is efficient, therefore nucleotides and RNA molecules must have emerged rapidly (< a few years) in order to avoid falling through pores at the base of the pond. We find that meteorites, not interplanetary dust particles, are the dominant source of nucleobases used for RNA synthesis. Finally, under these conditions, we find that first RNA polymers likely emerged before 4.17 Ga. / Thesis / Master of Science (MSc)
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Elucidating Evolutionary Mechanisms and Variants of the Hammerhead Ribozyme Using In Vitro SelectionBrill, Jake January 2024 (has links)
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.
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