The RNA World hypothesis describes a period of time during the origins of life in which RNA molecules performed all catalysis and were the only form of information storage. A great deal of evidence has been obtained in support of this hypothesis, however a few key demonstrations are lacking. The first demonstration is of a molecule capable of self-replication that could have plausibly arisen from the prebiotic soup. Previously in the Lehman Laboratory, a 198-nucleotide RNA was discovered that could be fragmented into as many as four pieces ranging from 39 - 63 nucleotides in length. When these pieces were incubated together in a test tube, they re-formed the necessary covalent bonds to regenerate the full-length 198-nucleotide RNA. Furthermore, the full-length RNAs were catalytically active and made copies of themselves from the remaining pieces in solution, providing a model system of self-replication. I was able to remove >10% of the total length of the RNA, which substantially reduced the catalytic activity of the full-length molecule. I discovered several mutations that restored catalytic activity by improved folding and increased catalytic rates using in vitro selection. A subset of these mutations was found to aid in the assembly of the shortened full-length RNA from smaller fragments than were possible in the original system, enhancing the prebiotic relevance of this system. A second demonstration to bolster the RNA World hypothesis would be showing that RNA is capable of harvesting energy from its environment by performing oxidation and reduction reactions. Again using in vitro selection, I have completed five rounds of selection geared towards identifying a ribozyme that reduces benzoic acid to benzaldehyde using Zn2+ and NADH. Results to date suggest the selection is working and it should be continued for another five to ten generations. Finally, I have discovered an RNA sequence that forms knots during transcription, a phenomenon heretofore undocumented in RNA. This new topology has implications for RNA stability by rendering RNA more resistant to hydrolysis, and could impact catalysis through formation of more complex, knotted active sites. Taken together, these findings have improved our understanding of RNA folding and catalysis, and the plausibility of the RNA World.

Long non-coding RNAs (lncRNAs) are often defined as transcripts >200nts that have no discernable protein-coding ability (Quinn and Chang, 2016). Although relatively little is understood about the molecular mechanisms of lncRNA function, they have established roles in regulation of gene expression during development, cell differentiation and pluripotency (Fatica and Bozzoni, 2014; Luo et al., 2016; Quinn and Chang, 2016; Rinn and Chang, 2012) across vastly diverse organisms ranging from plants to humans (Ulitsky and Bartel, 2013). LncRNAs have also been associated with numerous pathological conditions, such as cancers (Brunner et al., 2012), cardiovascular disease and neurodegeneration (Chen et al., 2013). Investigations into lncRNAs in wide ranging organisms, have revealed that many influence gene activity by forming ribonucleoprotein complexes that affect the conformational state of chromatin (Rinn and Chang, 2012). A genomic region that has revealed several functional lncRNAs in diverse organisms is the Hox complex (Pauli et al., 2011; Pettini, 2012; Rinn et al., 2007). The Hox complex encodes a set of transcription factors (TFs), physically clustered in the genome, which provide morphological identity along the anterior to posterior axis of developing embryos (Mallo and Alonso, 2013), throughout the majority of bilatarian animals (Moreno et al., 2011). Misexpression or mutation of Hox genes causes morphological and pathophysiological defects (Quinonez and Innis, 2014). We investigated clustering of lncRNAs throughout the D. melanogaster genome using available annotations and carried out RNA-seq in D. virilis to expand the repertoire of lncRNAs and identify clusters of lncRNAs. We found the Hox complex to be heavily enriched with lncRNAs in both organisms, and syntenic transcripts from D. melanogaster could be identified in D. pseudoobscura and D. virilis. Several lncRNAs aligned with polycomb response elements (PREs); transcription of PREs has previously been linked to a switch in their activity (Herzog et al., 2014). However, we found that transcribed PREs in D. melanogaster move positions relative to the protein-coding genes in other drosophilids, whilst the transcriptional units remain in the same syntenic region. Conservation of syntenic transcripts without evidence of remaining a PRE suggest that the transcription is not linked to PRE function, agreeing with recent findings that transcription of PREs does not affect their function (Kassis and Muller, 2015). We investigated functions of a novel lncRNA and adjacent PRE in the Hox complex by ectopic expression and utilization of other genetic manipulation tools. Overexpression of either the lncRNA or PRE and partial duplication of the lncRNA caused phenotypes such as missing halteres and/or T3 legs, misshaped T3 legs or malformed abdominal segments. The observations that ectopic expression of this lncRNA and an adjacent regulatory element from the Hox complex causes phenotypes that can be linked to adjacent Hox gene misregulation, Antp and Ubx, suggest that they are likely to have roles in the regulation of at least one of these Hox genes.

The emergence of life depended on the ability of the first biopolymer populations to thrive and approach larger population sizes and longer sequences. The evolution of these populations likely occurred under circumstances under which Muller's Ratchet in synergism with random drift could have caused large genetic deterioration of the biopolymers. This deterioration can drive the populations to extinction unless there is a mechanism to counteract it. The effect of the mutation rate and the effective population size on the time to extinction was tested on clonal populations of B16-19 ligase ribozymes, evolved with the continuous evolution in vitro system. Populations of 100, 300, 600 and/or 3000 molecules were evolved with and without the addition of Mn(II). The times to extinction for populations evolved without Mn(II) were found to be directly related to the effective size of the population. The small populations approached extinction at an average of 24.3 cycles; while the large populations did so at an average of 44.5 cycles. Genotypic characterization of the populations showed the presence of deleterious mutations in the small populations, which are the likely cause of their genetic deterioration and extinction via mutational meltdown. These deleterious mutations were not observed in the large populations; in contrast an advantageous mutant was present. Populations of 100 and 3000 molecules were evolved with Mn(II). None of the populations showed signs of genetic deterioration nor did they become extinct. Genotypic characterization of the 100-molecule population indicated the presence of a cloud of mutants closely genetically-related, forming a "quasispecies" structure.. The close connectedness of the mutants facilitates the recovery of one from another in the event of being removed from the population by random genetic drift. Thus, quasispecies shift the target of selection from individual to group. The total fitness of the molecules was measured by identifying the fitness component of the system that effect the ligase replication cycles: ligation, reverse transcription and transcription. It was found that the strength of the three components of fitness varied and that each one has a differential effect in the total absolute fitness of the ligases.

The secondary structure for nucleic acids provides a level of description that is both abstract enough to allow for efficient algorithms and realistic enough to provide a good approximate to the thermodynamic and kinetics properties of RNA structure formation. The secondary structure model has furthermore been successful in explaining salient features of RNA evolution in nature and in the test tube. In this contribution we review the computational chemistry of RNA secondary structures using a simplified algorithmic approach for explanation.

The origin of the first RNA polymers is central to most current theories regarding the origin of life. However, difficulties associated with the prebiotic formation of RNA have lead many researchers to conclude that simpler polymers, or proto-RNAs, preceded RNA. These earlier polymers would have been replaced by RNA over the course of evolution. A remaining difficulty for this theory is that the de novo synthesis of a feasible proto-RNA has not yet been demonstrated by plausible prebiotic reactions. This thesis focuses on two problems associated with prebiotic proto-RNA synthesis: The formation of nucleosides and the necessity of reversible backbone linkages for error correction in nucleic acid polymers. "The Nucleoside Problem", or the lack of success in forming pyrimidine nucleosides by plausible prebiotic reactions, represents a significant stumbling block to the RNA world hypothesis. Nearly four decades ago Orgel and coworkers demonstrated that the purine nucleosides adenosine and inosine are synthesized by heating and drying their respective bases and ribose in the presence of magnesium, but these reaction conditions do not yield the pyrimidine nucleosides uridine or cytidine from their respective bases. In this thesis a potential solution to The Nucleoside Problem is hypothesized based upon a proposed chemical mechanism for nucleoside formation. This hypothesis is supported by the successful synthesis of 2-pyrimidinone nucleosides by a plausible prebiotic reaction in good yield, demonstrating that pyrimidine nucleosides could have been available in the prebiotic chemical inventory, but that uridine and cytidine were likely not abundant. Reversible backbone linkages are necessary to provide a mechanism for error correction in non-enzymatic template-directed syntheses of proto-RNAs. In this thesis, acetals are explored as low-energy, reversible linkage groups for nucleosides in polymers. The synthesis of glyoxylate-acetal nucleic acids (gaNAs) through simple heating-drying reactions from neutral aqueous solutions is demonstrated, and these linkages are shown to be hydrolytically stable under a considerable range of solution conditions. Computational models demonstrate that the glyoxylate linkage is an excellent electronic and isosteric replacement for phosphate. Molecular dynamics simulations also indicate that a gaNA duplex would have structural properties that closely match a phosphate-linked RNA helix, suggesting the possibility for cross-pairing between gaNAs and RNAs, allowing for sequence transfer and genetic continuity through the evolution from proto-RNAs to RNA. The principles illustrated in this thesis by 2-pyrimidinone nucleoside and gaNA synthesis can be extended to other prebiotic condensation reactions. Factors affecting condensation yield, such as thermodynamics, kinetics, reactant solubility, and salt effects, are summarized herein.