Towards Building a Self-replicating Protocell: Nonenzymatic RNA Copying Driven by Potentially Prebiotic Activation and Higher-order Behaviors in Prebiotic Vesicles

Abstract

The emergence of a protocell capable of Darwinian evolution would require replicating compartments and genetic materials. Compartmentalization keeps informational polymers localized so that the functions they encode lead to an advantage in terms of replication or survival. Meanwhile, a mechanism for the inheritance of useful functions must exist. RNA and its close relatives are attractive candidate prebiotic molecules because they harbor the capacity for both inheritance and function in a single class of molecules. In this RNA world model, RNA polymers assemble from activated nucleotides, and when RNA oligomers of adequate lengths lead to ribozymes, RNA can begin to facilitate its own reproduction (Gilbert 1986). The propagation of functional RNAs within reproducing compartments leads to replicating protocells, ultimately giving rise to modern cellular life. My dissertation work explores both chemical and physical processes relevant to the transition from chemical to biological evolution on the early earth. The hypothesized central role of RNA in the origin of life suggests that RNA propagation predated the advent of complex protein enzymes. Such a process in turn requires a source of chemical energy for in situ nucleotide activation. Previously identified activation chemistries lead to damaging side reactions that destroy both templates and substrates (Biron and Pascal 2004, Fahrenbach, Giurgiu et al. 2017). A chemical process that can continuously reactivate hydrolyzed substrates and is compatible with chemical RNA copying is needed. A potentially prebiotic nucleotide activation that might be compatible with RNA copying remained elusive for nearly 60 years. I have demonstrated a potentially prebiotic pathway that chemically activates RNA nucleotides in a manner compatible with RNA copying (Zhang, Duzdevich et al. 2020). Following that, I have developed and characterized a prebiotically plausible scenario under which previously isolated key steps of nucleotide activation to nonenzymatic RNA copying can happen together under mutually compatible conditions (Zhang, Duzdevich et al. 2022). This pathway enables more realistic models of RNA propagation. Early cell membranes are thought to have been composed of fatty acids and related single-chain amphiphiles. Their probable involvement in the origin of life has been further demonstrated by their capability to grow and divide without complex biochemical machinery and encapsulate RNA templates that are being nonenzymatically copied (Hanczyc and Szostak 2004, Budin, Debnath et al. 2012, Adamala and Szostak 2013). However, without complex protein machinery, protocells would have had to rely on passive diffusion for internalizing nutrients. I have shown how primitive cells composed of fatty acids endocytose via a purely physicochemical process upon encountering nutrients in conjunction with extra membrane material. I then found that such inward vesiculation events could lead to internalization of nutrient solutes including mononucleotides and oligonucleotides, drawing further parallels to endocytosis. Such processes could have helped primitive cells capture nutrients that are otherwise impermeable. Taken together, these results demonstrate prebiotically plausible pathways from nucleotides to RNA copying as well as a scenario in which protocells internalize useful nutrients bypassing permeability limits, bringing us closer to developing primitive cells capable of Darwinian evolution.