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Amplification of RNA by an RNA polymerase ribozyme

Significance Darwinian life requires the ability to replicate genotypes and express phenotypes. Although all extant life relies on protein enzymes to accomplish these tasks, life in the ancestral RNA world would have used only RNA enzymes. Here, we report the in vitro evolution of an improved RNA polymerase ribozyme that is able to synthesize structured functional RNAs, including aptamers and ribozymes, and replicate short RNA sequences in a protein-free form of the PCR. Thus, the replication of RNA and the expression of functional RNA can be accomplished with RNA alone. Combining and improving these activities may enable the self-sustained evolution of RNA and offers a potential route to a synthetic form of RNA life. In all extant life, genetic information is stored in nucleic acids that are replicated by polymerase proteins. In the hypothesized RNA world, before 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 is able to synthesize a variety of complex structured RNAs, including aptamers, ribozymes, and, in low yield, even tRNA. Furthermore, the polymerase can replicate nucleic acids, amplifying short RNA templates by more than 10,000-fold in an RNA-catalyzed form of the PCR. 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.