Date of Award

Spring 2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular, Cellular, and Developmental Biology

First Advisor

Breaker, Ronald

Abstract

The RNA World theory postulates that at a certain point around 4 billion years ago during an RNA World period, a molecule, possibly RNA, developed the ability to self-replicate via polymerization. The theory also suggests that all extant life evolved from this initial RNA-based predecessor. No geological evidence from this RNA World period remains, and a self-replicating RNA polymerase has yet to be discovered or created. However, there is ample evidence from extant life pointing to an RNA-based predecessor. For example, the ribosome is a ribozyme, RNA functions as precursor signaling molecules, riboswitches regulate transcription and translation, and many more. For billions of years and even today, natural selection and the physical laws of nature direct and select molecules deemed fit for the pressure present in their environment. In this way, RNA has been evolving in darkness, unwitnessed by the very entities that are comprised of and still in part regulated by it. With advancements in next-generation sequencing and nucleic acid selections, scientists now witness novel RNA functions that sometimes are accompanied by large structural changes to the RNA secondary structure. Using techniques like in vitro and in vivo selections, we can now control the selection pressure on these molecules. We can now evaluate selections with next-generation sequencing and machine learning to witness RNA evolution in action.      In the past thirty years, scientists have made major contributions towards the selection of a self-replicating polymerase molecule. In 1995, scientists used in vitro selections to create the class I ligase, an RNA that could join two separate strands of RNA through covalent phosphodiester linking. The ligase was selected from a pool of random RNA sequences, much like there would have been on the early earth during this RNA World period. Through further selection, the class I ligase evolved into the molecule termed R18, which had the ability to polymerize from an RNA primer on an RNA template. From the R18 molecule, multiple research groups developed branching RNA polymerase ribozyme lineages, all with the common goal of selecting for a self-replicating molecule. In addition to these branching lineages, non-enzymatic assembly of polynucleotides has also been developed. Despite the significant effort placed in selecting for a self-replicating RNA, such a molecule remains elusive. To understand the role that RNA evolution has played in the development of extant life, we must first understand how RNA evolved to encompass all the roles it serves in the multitude of functions in life today. My colleagues designed a modified version of the R18 polymerase ribozyme, deemed “WT,” which served as the starting sequence for their selections. To evolve the WT polymerase, they developed selection strategies that utilized functional RNAs such as aptamers and self-cleaving ribozymes. They then carried out 52 rounds of either aptamer or self-cleaver selections on this WT population. Every few rounds, a small number of polymerase variants were cloned out of the evolved population. An even smaller number of polymerase variants were biochemically validated to determine if they had increased in polymerization rate. Rather than validating a few cloned sequences, I developed a bioinformatic pipeline that resulted in the ability to tally, align, and cluster all variant sequences in a given selection population. I used the bioinformatic pipeline on every few rounds of selection within the WT lineage, tallying and tracking the frequency of RNA sequence variations over 52 rounds of selection. I then used this method to validate a novel RNA secondary structure pseudoknot rearrangement, termed P8, in the polymerase population.       I subsequently validated the novel secondary structure rearrangement by using in line probing, an in vitro biochemical technique used to determine an RNA’s secondary structure or interaction with another molecule or ligand. The secondary structure for six variant sequences that were pulled from the 52 rounds of selection allowed us to witness how the novel secondary structure pseudoknot gradually evolved to a greater fitness peak. Ribozymes, in particular the RNA polymerase ribozyme, are thought to occupy high and isolated fitness peaks that are tied to the molecule’s secondary structural elements. Because these secondary structural elements are tightly associated with the ribozyme’s optimized fitness peak, exploring alternative structures generally leads to severe negative consequences for fitness. With the bioinformatics pipeline mentioned above, I clustered highly represented variants by the sequence of their P8 region. The P8 pseudoknot structure spontaneously emerged during the evolution process and was optimized and conserved after 28 rounds of selection. Next, I transplanted the novel P8 pseudoknot from the 52-2 variant into the WT sequence. The results of that experiment show that the P8 was necessary, but not sufficient to improve the WT catalytic activity to the 52-2 variant’s capacity. The results showed that the novel P8 region was indeed a jump to a higher fitness local. To my knowledge and after thorough analysis of the literature of the field, this is the first RNA secondary structure remodeling that has been validated and witnessed mid-evolution in a synthetically evolved RNA. Additionally, no such secondary structure remodeling of a natural RNA has been observed. Witnessing the evolution of RNA either synthetic or from nature provides a powerful means of control and understanding our RNA ancestors, our current RNA components, and any future RNA evolution target we select.       Contained within this document I provide a review of instances where RNA evolution has been witnessed, starting 4 billion years ago following the proposed end of an RNA World transition from RNA- to DNA-protein based life, to the present time. Advances in in vitro/vivo selections and next-generation sequencing reveal RNA evolution in action today. Described are instances where scientists have witnessed natural RNA evolution and synthetic RNA evolution, providing evidence for a prehistoric RNA World and a path forward for future RNA evolution advancements. From this breadth of literature, it would appear that the RNA World continues today.      Following this review, I outline my discovery of an RNA polymerase ribozyme that underwent the first observed structural rearrangement of a synthetic RNA, which resulted in an increase in its activity. Furthermore, the RNA polymerase can now synthesize a full length, active copy of its ancestral molecule the class I ligase. While there are other examples of RNA polymerase lineages from other research groups that are mid-evolution, this lineage that I present is the first to catalog a structural rearrangement. I developed bioinformatic means to track the evolution of the RNA polymerase ribozyme. This bioinformatic pipeline can be developed further to track any synthetic or natural RNA evolution over many generations and it provides the foundation to work toward a self-replicating RNA by enabling scientists to design more informed selections. The inevitable discovery of a self-replicating RNA will serve as incontrovertible evidence that RNA has the capacity to initiate darwinian evolution and may demonstrate a possible route to the discovery of the origin of life as we understand it on earth.

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