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)

One of the greatest scientific mysteries of all time is the Origin of Life on Earth. Life on Earth may have emerged with a unique molecule: Ribonucleic Acids (RNA). The RNA world for the origin of life is a theory that states that life started with RNA before DNA and proteins because RNA molecules can auto-replicate and store genetic information. This thesis aims to expose how such RNA molecules could have been formed on a primitive Earth without the presence of other catalytic biomolecules such as enzymes. The model used in this thesis is the warm little ponds theory for the origin of life. RNA molecules could have been formed in these ponds thanks to wet-cold and warm-dry cycles. We used new experimental and computational technologies to try to answer this dilemma. Using a new machine, the Planet Simulator, which can mimic primitive environments by controlling five physical parameters, we found that extreme heat and low pH would destroy the building blocks of RNA. However, Molecular Dynamics computer simulations showed us that neutral pH could have led to the formation of RNA. Still, the presence of any surfaces and substrates would have decreased the polymerization rate due to the number of interactions between the RNA building blocks and the minerals substrates. We then found a new vision of where life could have come from: in super-saturated water droplets, which could have been formed by geysers or springs on primitive Earth. We tested this theory experimentally using an acoustic levitator to levitate super-saturated droplets and study them in the laboratory. Our preliminary results showed that RNA could have been formed in such droplets on primitive Earth. / Thesis / Master of Science (MSc)

Metabolism is a key attribute of life on Earth at multiple spatial and temporal scales, involved in processes ranging from cellular reproduction to biogeochemical cycles. While metabolic network modeling approaches have enabled significant progress at the cellular-scale, extending these techniques to address questions at both the ecosystem and planetary-scales remains highly unexplored. In this thesis, I integrate various multi-scale metabolic network modeling approaches to address key questions with regard to both the long-term evolution of metabolism in the biosphere and the metabolic processes that take place in complex microbial communities. The first portion of my thesis work, focused on the evolution of ancient metabolic networks, attempts to model the emergence of ecosystem-level metabolism from simple geochemical precursors. By integrating network-based algorithms, physiochemical constraints, and geochemical estimates of ancient Earth, I explored whether a complex metabolic network could have emerged without phosphate, a key molecular component in modern-day living systems, known to be poorly available at the onset of life. We found that phosphate may have not been essential in early living systems, and that thioesters may have been the primitive energy currency in ancient metabolic networks. By generalizing this approach to explore the scope of geochemical scenarios that could have given rise to living systems, I found that other key biomolecules, including fixed nitrogen, may have not been required at the earliest stages in biochemical evolution. The second portion of my thesis deals with a different aspect of ecosystem-level metabolism, namely the role of metabolism in shaping the structure of microbial communities. I studied the relationship between metabolism and microbial community assembly using microbial communities grown in synthetic laboratory environments. We found that a generalized statistical consumer-resource model recapitulates the emergent phenomena observed in these experiments. Future work could seek to better clarify the connection between the fundamental rules that led to life’s emergence over 4 billion years ago and the laws that shape microbial ecosystems today. An ecosystems-level metabolic perspective may aid in our understanding of both the emergence and maintenance of the biosphere.

Earth’s crust and primordial ocean formed more than 4 billion years ago and life is believed to have originated on earth at least 3.6 billion years ago. This suggests that primitive cellular life must have evolved from non-living matter during that period of several hundred million years. To study the transition from chemistry to biology, a simple vesicular system called a protocell is an ideal model that is self-organized and contains informational or metabolic materials. This thesis starts by exploring the replication of a model genetic material under plausible prebiotic conditions. The non-enzymatic copying of RNA was found to be catalyzed by Fe2+, which used to be abundant in aqueous environments on the early anoxic earth. Fe2+ was found to be a better catalyst of non-enzymatic RNA copying and ligation in slightly acidic to neutral pH conditions than Mg2+, the divalent cation used to catalyze these reactions in previous studies. This finding suggests that ferrous iron could have facilitated the replication and evolution of RNA on the prebiotic earth. To gain a better understanding of the properties of protocell membranes, the impact of membrane composition and multi-bilayer structure on non-enzymatic and enzymatic biochemical reactions was studied. A fatty acid/phospholipid hybrid membrane system was proposed as a potential intermediate state in protocellular evolution. This membrane composition was investigated for its stability and permeability, two fundamental features of functional protocells. The system proved stable in the presence of divalent cations and retained permeability to small building block molecule. Vesicles with this composition were shown to host faster non-enzymatic RNA copying, and to enable enzymatic protein synthesis. To study the effects of multi-lamellarity, giant multilamellar vesicles (GMVs) were prepared by an extrusion-dialysis method. Compared with small unilamellar vesicles (SUVs), GMVs show slightly better ability to retain encapsulated RNA, while maintaining good permeability for small charged molecules. The multilamellar structure also promotes non-enzymatic RNA copying, providing preliminary evidence that membranes could also mediate catalytic functions as well as acting as a compartment. / 2020-07-02T00:00:00Z

Modeling chemical environments is an important step to understanding the diversity of prebiotic systems that may have formed on the early earth or potentially can occur on other worlds. By using the modern Earth as a test case, these models predict scenarios with systems more conducive to the formation of the organic molecules that are important to life. Here we use the equilibrium thermodynamic modeling program HSC Chem to investigate prebiotic environments. This program uses the raw material that the user inputs into the system in order to calculate the change in amounts of chemical species forming as a function of temperature and pressure using equilibrium (batch reactor) chemistry. Our results show that that ferrous ion (Fe2+), which may be important in the early formation of organic molecules on Earth, is most abundant in the aqueous phase where the atmosphere contains carbon dioxide as a major constituent. A pure methane atmosphere exhibits the lowest concentrations of this ion, and mixtures tend to end up in between the two extremes. Additionally, we have determined the pH of early oceans, which has implications for biomineralization, chemical reactions, and mineral chemistry. We see that the CO2 atmosphere, and to some extent, the mixtures and CH4 atmospheres, exhibit near neutral pHs. These results allow prediction of processes that might have taken place and could have impacted the development of life on the early earth.

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.

<p> Popular theories about the origin of life can be classified to two classes: metabolism first or genes first. As a metabolism first theory, the lipid world theory, in which non-covalent assemblies of lipids, such as micelles and vesicles store information in the form of a non-random molecular composition, has been proposed to investigate the possibility of inheritance without genes. Our models assume that interaction occurs between nearest neighbour molecules only, and account for spatial segregation of molecules of different types within the assembly. We also draw a distinction between a self-assembly model, in which the composition is determined by mutually favourable interaction energies between the molecules, and a catalytic model, in which the composition is determined by mutually favourable catalysis. We show that compositional inheritance occurs in both models, although the self-assembly case seems more relevant if the molecules are simple lipids. In the case where the assemblies are composed of just two types of molecules, there is a strong analogy with the classic two-allele Moran model from population genetics. This highlights the parallel between compositional inheritance and genetic inheritance. We also investigated the polymerization reactions which may bridge the gap between simple organic molecules and the beginning of the RNA world, which belongs to the class of genes first theories. We found that different from normal chemical systems, catalysts for the polymerization system can shift the equilibrium toward longer polymers. Together with RNA's potential as catalyst, the RNA polymerization system may form a feedback loop which makes the formation of functional RNA molecules easier, and come more close to the beginning of RNA world.</p> / Thesis / Master of Science (MSc)

Esta pesquisa apresenta os resultados de um levantamento empírico de livros didáticos de Biologia e Química aprovados no PNLEM 2007, no sentido de investigar as demandas de conceitos químicos no estudo de uma temática própria da disciplina de Biologia: a origem da vida. Adicionalmente, esta dissertação coteja essas demandas conceituais com os correspondentes saberes químicos sequenciados nos capítulos das coleções de Química e discute a potencial interlocução entre os conjuntos de saberes das duas disciplinas, visto que fazem parte da mesma área do conhecimento escolar. Considerando-se todas as obras divididas em três volumes um para cada ano do Ensino Médio e excetuando-se os volumes únicos, foram analisados os capítulos que tratam da origem da vida em quatro coleções de Biologia e todo o conteúdo programático de duas obras de Química. Reconhecendo a relevância do livro didático no cenário educacional brasileiro, o caráter notadamente disciplinar do currículo e as especificidades do ensino de Ciências, este trabalho reúne argumentos teóricos que fundamentam a necessidade de um olhar abrangente sobre a realidade, sempre complexa e multifacetada. / This research presents the results of an empirical survey from textbooks of Biology and Chemistry approved in PNLEM 2007, to investigate the demands of chemical concepts in the study of one the subjects in Biology: the origin of life. In addition to that, this dissertation collates these conceptual demands with the corresponding chemical knowledge sequenced in chapters of the chemical collections and discusses the potential dialogue between the sets of knowledge of those two disciplines, as part of the same area of school knowledge. Considering the works divided into three volumes one for each year of high school and except for the single volumes, were analyzed the chapters dealing with the origin of life in four collections of Biology and whole academic program in two works of Chemistry. Recognizing the relevance of the textbook in Brazilian educational scenario, the notably disciplinary character of the curriculum and the specificities of Natural Sciences who originated the school disciplines of Biology and Chemistry, this work gathers theoretical arguments that justify the need for a comprehensive look at the reality, always complex and multifaceted.

The RNA World hypothesis suggests that RNA, or a proto-RNA, existed in an early form of life that had not yet developed the ability to synthesize protein enzymes. This hypothesis, by some interpretations, implies that nucleic acid polymers were the first polymers of life, and must have therefore spontaneously formed from simple molecular building blocks in the "prebiotic soup." Although prebiotic chemists have searched for decades for a process by which RNA can be made from plausible prebiotic reactions, numerous problems persist that stand in the way of a chemically-sound model for the spontaneous generation of an RNA World (e.g., strand-cyclization, heterogeneous backbones, non-selective ligation of activated nucleotides). The Molecular Midwife hypothesis, proposed by Hud and Anet in 2000, provides a possible solution to several problems associated with the assembly of the first nucleic acids. In this hypothesis, nucleic acid base pairs are assembled by small, planar molecules that resemble molecules which are known today to intercalate the base pairs of nucleic acid duplexes. Thus, the validity and merits of the Molecular Midwife hypothesis can be, to some extent, explored by studying the effects of intercalation on the non-covalent assembly of nucleic acids. In this thesis, I explore the role of the sugar-phosphate backbone in dictating the structure and thermodynamics of nucleic acid intercalation by using 2′,5′-linked RNA intercalation as a model system of non-natural nucleic acid intercalation. The solution structure of an intercalator-bound 2′,5′ RNA duplex reveals structural and thermodynamic aspects of intercalation that provide insight into the origin of the nearest-neighbor exclusion principle, a principle that is uniformly obeyed upon the intercalation of natural (i.e. 3′,5′-linked) RNA and DNA. I also demonstrate the ability of intercalator-mediated assembly to circumvent the strand-cyclization problem, a problem that otherwise greatly limits the polymerization of short oligonucleotides into long polymers. Together, the data presented in this thesis illustrate the important role that the nucleic acid backbone plays in governing the thermodynamics of intercalation, and provide support for the proposed role of intercalator-mediated assembly in the prebiotic formation of nucleic acids.

Syftet med studien är att synliggöra hur barn tänker om liv och död och deras uppfattning om livets uppkomst. Genom att utgå från ett biologiskt- och ett filosofiskt perspektiv så är mitt syfte att synliggöra barns oliktänkande i ämnet. Min valda metod till studien är semistrukturerad intervju integrerat med bildskapande med barn mellan 5-7 år. I resultatet av min studie så synliggörs barns olika sätt att förstå och uttrycka livsnära fenomen på. Resultatet visade också på barns oliktänkande genom en naturalistisk-, evolutionär- eller en skapelsekopplad beskrivning av livets och människans uppkomst. De medverkande barnen visade en nyfikenhet, engagemang och en tydlig vilja till att reflektera i givna ämnen. Den insamlade data utgjorde en tydlig bild av barns oliktänkande runt begreppen levande, liv och död och livets uppkomst. / The main purpose of this study is to make children’s thoughts about life, death and their concept of origin of life visible. Through a combined biological- and philosophical perspective my aim is to make children’s diversity of thoughts visual. My chosen method for my study is semi-structured interviews integrated with drawings with children between 5-7 years old. In the result of the study children´s different way of understanding and expressing their thoughts about life become visible. The result also shows children’s different concept about origin of life connected to a naturalistic-, an evolutionary -and a creationist theory. I noticed a great curiosity and engagement from the participating children and a distinct commitment to reflect over given topics. The collected data gave a clear illustration of children’s different kind of thoughts of being alive, life and death and their understanding of origin of life.