RNA Chemistry and the RNA World

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The origin of life is a fundamental problem in biology. The presence of RNA at the core of the ribosome, the extensive use of nucleotide cofactors, and the synthesis of DNA and some amino acids from RNA precursors suggests that RNA was once the dominant biological catalyst and genetic polymer. This `RNA World' hypothesis assumes that RNA is capable of all the reactions required to maintain and evolve a `ribo-organism'. One of my laboratory's long-term objectives is to isolate an RNA replicase ribozyme capable of copying and transcribing its own genome and thus enabling self-sustaining RNA based evolution in the laboratory. Such a system could be called `alive' and would marry the underlying simplicity of chemistry to the complexity of biology. This laboratory-based understanding of evolution would inform, as never before, about the dawn of life on this planet. Such an ambitious endeavour must be approached in stages. Semi-conservative replication requires a polymerase able to convert single-stranded template processively into a duplex product. In my previous Discovery grant, I proposed and then developed a promoter-dependent RNA polymerase built entirely from RNA. This ribozyme can form a topological clamp around a single-stranded RNA template and then fill it in to RNA duplex (Cojocaru and Unrau, Science 2021). Such topological clamps are commonly found in protein polymerases, but what is still needed to enable RNA replication by RNA? Processivity and strand displacement are tightly coupled in extant genomic replication, but how could the mechanism of strand displacement have evolved early in evolution? My laboratory will evolve our existing RNA polymerase ribozyme into one able to invade RNA duplex. RNA rolling circle replication could have played a fundamental role early in evolution. Indeed, if one replaces the RNA in rolling circle replication with the DNA in a replication fork, the two replicative processes look identical: A polymerase invades downstream duplex copying the leading single-strand into duplex while simultaneously liberating the lagging-strand. Three aims will explore this complex mechanism from an evolutionary perspective: I. Increasing the Processivity of the Clamping Polymerase Ribozyme by Stringent Topological Selection. The processivity of our topologically clamping RNA polymerase ribozyme will be improved using a new in vitro selection approach. II. Direct Selection for Ribozyme Polymerases able to Perform Strand Displacement. A thermal ratchet will be evolved to facilitate downstream strand invasion. III. Selection for translocating poly(A) polymerase ribozymes. A ribozyme polymerase that uses a `power stroke' to translocate and that is mechanistically similar to extant protein polymerases will be selected. This work will develop cutting edge Canadian RNA biochemical methods, train HQP, and is of global significance to understanding the evolution of life on this planet.