At some point in the origin of life, life likely went through a stage where RNA acted as genome and catalyst-- the RNA world. Eventually, life evolved into the form we observe today, with DNA acting as genome and proteins acting as the main catalysts. Although there is no known direct evidence of RNA world organisms, we can gain understanding of life's early ancestors by creating catalytic RNAs in the lab that could have existed in an RNA world. This dissertation aims to develop and improve two of these catalytic RNAs (ribozymes). The first is a triphosphorylation ribozyme, a catalytic RNA that triphosphorylates the 5' hydroxyl groups of RNA using trimetaphosphate. In a RNA world, this activity could have been useful for chemically activating RNAs for ligation or polymerization. The second is a polymerase ribozyme, a catalytic RNA that catalyzes RNA polymerization. This activity could have been important for replication in a RNA world organism. In order to identify catalytic RNAs that can triphosphorylate their own 5' hydroxyl groups, an in vitro selection method was established. From a library of ~10¹⁴ random sequences, several active sequences were identified and one was analyzed in greater detail. This triphosphorylation ribozyme was modified to act in trans and had a reaction rate of 0.16 min⁻¹ under optimal conditions. Preliminary analysis of its secondary structure suggests that it forms a four helical junction motif. A catalytic RNA that catalyzes the polymerization of RNA was previously developed, but it is too inefficient to replicate itself. Its limitation is weak substrate binding. Here efforts were made to improve the polymerase ribozyme by reducing charge repulsion between the negatively charged ribozyme and its substrate using positively charged amino acids as cofactors. However, these cofactors did not improve ribozyme polymerization. An in vitro selection method that puts selection pressure on the ribozyme to bind its substrate in trans was developed to try to find improved polymerase ribozymes. Although no active ribozymes were found, several steps of a technically challenging in vitro selection method were established. In summary, this thesis presents work to develop two catalytic RNAs for a RNA world. An existing polymerase ribozyme was tested with amino acid cofactors and progress was made towards developing an in vitro selection that could be used to find novel polymerase ribozymes. Also, a novel catalytic RNA that can triphoshphorylate RNAs was created. This opens up new avenues that will deepen our understanding of the RNA world

The RNA world hypothesis describes a stage in the early evolution of life in which RNA served as genome and as the only genome-encoded catalyst. To test whether RNA world organisms could have used cyclic trimetaphosphate as an energy source, we developed an in vitro selection strategy for isolating ribozymes that catalyze the triphosphorylation of RNA 5'-hydroxyl groups with trimetaphosphate. Several active sequences were isolated, and one ribozyme was analyzed in more detail. The ribozyme was truncated to 96 nt, while retaining full activity. It was converted to a trans-format and reacted with rates of 0.16 min(-1) under optimal conditions. The secondary structure appears to contain a four-helical junction motif. This study showed that ribozymes can use trimetaphosphate to triphosphorylate RNA 5'-hydroxyl groups and suggested that RNA world organisms could have used trimetaphosphate as their energy source.

The emergence of catalytic RNA is believed to have been a key event during the origin of life. Understanding how catalytic activity is distributed across random sequences is fundamental to estimating the probability that catalytic sequences would emerge. Here, we analyze the in vitro evolution of triphosphorylating ribozymes and translate their fitnesses into absolute estimates of catalytic activity for hundreds of ribozyme families. The analysis efficiently identified highly active ribozymes and estimated catalytic activity with good accuracy. The evolutionary dynamics follow Fisher's Fundamental Theorem of Natural Selection and a corollary, permitting retrospective inference of the distribution of fitness and activity in the random sequence pool for the first time. The frequency distribution of rate constants appears to be log-normal, with a surprisingly steep dropoff at higher activity, consistent with a mechanism for the emergence of activity as the product of many independent contributions.

The RNA world hypothesis states that the early evolution of life went through a stage in which RNA served both as genome and as catalyst. The central catalyst in an RNA world organism would have been a ribozyme that catalyzed RNA polymerization to facilitate self-replication. An RNA polymerase ribozyme was developed previously in the lab but it is not efficient enough for self-replication. The factor that limits its polymerization efficiency is its weak sequence-independent binding of the primer/template substrate. Here we tested whether RNA polymerization could be improved by a cationic arginine cofactor, to improve the interaction with the substrate. In an RNA world, amino acid-nucleic acid conjugates could have facilitated the emergence of the translation apparatus and the transition to an RNP world. We chose the amino acid arginine for our study because this is the amino acid most adept to interact with RNA. An arginine cofactor was positioned at ten different sites on the ribozyme, using conjugates of arginine with short DNA or RNA oligonucleotides. However, polymerization efficiency was not increased in any of the ten positions. In five of the ten positions the arginine reduced or modulated polymerization efficiency, which gives insight into the substrate-binding site on the ribozyme. These results suggest that the existing polymerase ribozyme is not well suited to using an arginine cofactor.