Publication Date

8-1-2025

Date of Final Oral Examination (Defense)

4-18-2025

Type of Culminating Activity

Dissertation

Degree Title

Doctor of Philosophy in Biomolecular Sciences

Department

Biological Sciences

Supervisory Committee Chair

Eric Hayden, Ph.D.

Supervisory Committee Member

Juliette Tinker, Ph.D.

Supervisory Committee Member

Daniel Fologea, Ph.D.

Abstract

The RNA World is the theory that life on Earth originated as RNA molecules able to both carry genetic information and catalyze enzymatic activities, without the need of DNA nor proteins. Abundant evidence supports that RNA preceded the emergence of DNA and proteins, the most notable being that both DNA replication and protein synthesis require RNA components. It has been hypothesized that the RNA World originated as a self-replicating RNA system. Furthermore, since the activity of modern-day catalytic RNA (ribozymes) can be affected by reaction conditions (e.g. Mg^2+ cofactor concentrations), it is hypothesized that evolution in the RNA World may have been steered by such environmental conditions.

Decades of experiments have shown how RNA subcomponents (e.g. nucleobases and sugars) may have formed under prebiotic conditions and that short RNA oligonucleotides may spontaneously polymerize from rNTPs. The mechanism bridging the gap between short inactive oligonucleotides and complex ribozymes could be explained by covalent self-constructing ribozymes: small inactive RNA oligonucleotides capable of spontaneously constructing a more complex full-length ribozyme. Unfortunately, only one example of such ribozyme has been documented , so much more exploration of self-constructing RNA is needed.

The first chapter of this dissertation introduces the Phormidium ribozyme and demonstrates its ability to covalently self-construct from smaller inactive RNA fragments. The first chapter also explores the sequence space surrounding the Phormidium ribozyme and identified 24,678 of its variants that are able to covalently self-construct to produce the full-length catalytic RNA molecule. This supports the idea that the complexity necessary for a self-replicating RNA system may have emerged from a prebiotic environment by the cooperation of small inactive RNA oligonucleotides.

Environmental effects on the evolution of the RNA World have not been adequately explored. The catalytic activity of RNA populations at the origin of life would have likely been subject to random mutations. Some mutations may have provided mutated RNA with an advantage over the rest of the population, allowing beneficial mutations to accumulate, thus driving the evolution of RNA populations. Different environmental conditions would have influenced the effect of random mutations on whether these were advantageous or not. The synergistic effects of mutations and environmental conditions in the RNA World have hardly been explored.

The second chapter of this dissertation investigates how environmental changes may have steered evolution during the RNA World. The environmental variable of this study was the concentration of magnesium ions, which are essential for stabilizing the structure of group I intron ribozymes. For this study a library of 16,384 variants of the Azoarcus group I intron was used to observe their molecular fitness at different concentrations of magnesium. Fitness values were plotted into fitness landscapes that were later used to computer simulate evolution in different scenarios. Overall, changes in the magnesium concentration in the environment were observed to affect the ruggedness of an RNA fitness landscape and consequently influence evolution. Furthermore, an improvement of the Azoarcus group I intron fitness landscape was attempted by using a kinetic approach.

Comments

Gianluca Peri, ORCID: 0000-0003-4559-7341

DOI

https://doi.org/10.18122/td.2427.boisestate

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