In all extant life, genetic information is stored in nucleic acids that are replicated by polymerase proteins. In the hypothesized RNA world, prior to the evolution of genetically-encoded proteins, ancestral organisms contained RNA genes that were replicated by an RNA polymerase ribozyme. In an effort toward reconstructing RNA-based life in the laboratory, in vitro evolution was used to improve dramatically the activity and generality of an RNA polymerase ribozyme by selecting variants that can synthesize functional RNA molecules from an RNA template. The improved polymerase ribozyme can synthesize a variety of complex structured RNAs, including tRNA and component fragments of itself. Furthermore, the polymerase can replicate nucleic acids, amplifying short RNA templates by more than 10,000-fold in an RNA-catalyzed form of the polymerase chain reaction (riboPCR). Thus the two prerequisites of Darwinian life — the replication of genetic information and its conversion into functional molecules — can now be accomplished with RNA in the complete absence of proteins. The polymerase also shows broad catalytic promiscuity — transcribing RNA templates into strands of deoxyribo-, threo-, or arabino- nucleic acids (DNA, TNA, and ANA) and DNA or TNA templates back into RNA. It even shows modest activity as a DNA-templated DNA polymerase. Thus evolutionary transitions between genetic systems of different structures before the invention of proteins, including that between the RNA world to the modern DNA-based world, may have been readily accessible. Ongoing in vitro evolution to synthesize complete functional ribozymes within minutes may achieve fully autonomous RNA replication of the polymerase itself and other ribozymes of similar complexity. Such a general RNA replicase could, under appropriate conditions, achieve self-sustained Darwinian evolution and would arguably constitute a synthetic form of RNA life.

Toward RNA life: Synthesis and replication of RNA by RNA

255th National Meeting (2018)

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