In Search of Primordial RNA: The Effects of Noncanonical Nucleotides on Nonenzymatic Primer Extension

Abstract

The observation that the cellular machinery comprised of DNA, RNA, and proteins is more or less preserved across all terrestrial life evokes the thought that all life may stem from a unified origin. One of the many theories for biogenesis, the “RNA World” hypothesis posits that RNA was the first biopolymer that carried out all cellular function responsible for maintaining and propagating life. In early biological systems, RNA would serve a dual role in genetic information storage and transfer (allowing for inheritance) and as a catalyst of the necessary functions for sustaining life. This hypothesis is supported by RNA’s diverse functions in modern biology, as a messenger of genetic information from DNA to proteins, and as a vital catalyst as the ribozyme in the ribosome, as well as by the observation that RNA serves a vestigial role in many of the most important and ubiquitous co-factors. However, there are many unanswered questions concerning the progression from a completely chemical world to the first forms of RNA life. In Chapter 1, I discuss the key requirements for the RNA world hypothesis to hold true. The first key requirement for RNA to emerge as the primordial biopolymer is the abiotic synthesis of ribonucleotide monomers; I cover the progression from historical to modern syntheses. Satisfactory syntheses of pyrimidine ribonucleosides have been accomplished, but purine ribonucleosides remain a problem, with low-yielding routes that produce byproducts. Assuming all ribonucleotide monomers can be made and subsequently polymerize to form random sequence RNA, the next key problem is the ability for RNA to propagate through nonenzymatic copying. Thus, I then review the state of the art of nonenzymatic template-directed copying of RNA, focusing on rates and fidelity of informational inheritance as readouts for success. Given the wealth of byproducts produced in prebiotic ribonucleotide synthesis, coupled with the lack of a purine synthesis, evaluating the behavior of non-canonical nucleotides in RNA copying is quintessential. Recently a prebiotic pathway to 8-oxo-adenosine and 8-oxo-inosine has been described, raising the question of the suitability of 8-oxo-purines as substrates for prebiotic RNA replication. In Chapter 2, I demonstrate that 8-oxo-purine nucleotides are poor substrates for nonenzymatic RNA primer extension; both the rate and fidelity of primer extension are poor. On the other hand, inosine exhibits surprisingly rapid and accurate nonenzymatic RNA copying. Inosine, which can be derived from adenosine by deamination, exhibits copying properties consistent with the hypothesis that it could have replaced guanosine in the earliest stages of the emergence of life. Recent prebiotic synthetic routes suggest ribonucleotide synthesis may have been accompanied by the synthesis of arabino- and 2′-deoxyribonucleotides. To determine how relatively homogeneous RNA could have emerged from complex mixtures, I discuss the properties of arabino- and 2′-deoxyribonucleotides in nonenzymatic template-directed primer extension reactions in Chapter 3. While arabino-, and to a lesser extent 2′-deoxyribonucleotides, do not possess copying profiles consistent with a main role in the primordial genetic polymer, experiments with mixtures of nucleotides suggest that the coexistence of ribo- and arabino- nucleotides does not impede the copying of RNA templates. Moreover, chimeric oligoribonucleotides containing 2′-deoxy- or arabinonucleotides act as efficient templates for RNA copying. Thus, one can envision a scenario in which the initial genetic polymers were random sequence chimeric oligonucleotides formed by untemplated polymerization, but that template copying chemistry favored RNA synthesis; multiple rounds of replication may have led to pools of oligomers composed mainly of RNA.