Structural transition of replicable RNAs during in vitro evolution with Qβ replicase.

Ryo Mizuuchi,Kimihito Usui,Norikazu Ichihashi

doi:10.1261/rna.073106.119

Ryo Mizuuchi, Kimihito Usui + Show 1 more

Open Access

https://doi.org/10.1261/rna.073106.119

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Abstract

Single-stranded RNAs (ssRNAs) are utilized as genomes in some viruses and also in experimental models of ancient life-forms, owing to their simplicity. One of the largest problems for ssRNA replication is the formation of double-stranded RNA (dsRNA), a dead-end product for ssRNA replication. A possible strategy to avoid dsRNA formation is to create strong intramolecular secondary structures of ssRNA. To design ssRNAs that efficiently replicate by Qβ replicase with minimum dsRNA formation, we previously proposed the “fewer unpaired GC rule.” According to this rule, ssRNAs that have fewer unpaired G and C bases in the secondary structure should efficiently replicate with less dsRNA formation. However, the validity of this rule still needs to be examined, especially for longer ssRNAs. Here, we analyze nine long ssRNAs that successively appeared during an in vitro evolution of replicable ssRNA by Qβ replicase and examine whether this rule can explain the structural transitions of the RNAs. We found that these ssRNAs improved their template abilities step-by-step with decreasing dsRNA formation as mutations accumulated. We then examine the secondary structures of all the RNAs by a chemical modification method. The analysis of the structures revealed that the probabilities of unpaired G and C bases tended to decrease gradually in the course of evolution. The decreases were caused by the local structural changes around the mutation sites in most of the cases. These results support the validity of the “fewer unpaired GC rule” to efficiently design replicable ssRNAs by Qβ replicase, useful for more complex ssRNA replication systems.

Highlights

The replication of genetic information is a fundamental function of living systems
A possible method to overcome this problem is to use Single-stranded RNA (ssRNA) that is likely to form intramolecular base-pairings, which hinder intermolecular base-pairings. The design of such an ssRNA sequence is possible by making secondary structures throughout the sequence and decreasing unpaired G and C nucleotides, which facilitate the association of the ssRNA with the complementary strand (Usui et al 2015)
We performed an in vitro evolutionary experiment using ssRNA (2125 nt) that encoded the catalytic subunit of Qβ replicase in a reconstituted translation system of Escherichia coli (Shimizu et al 2001; Ichihashi et al 2013)

Summary

INTRODUCTION

The replication of genetic information is a fundamental function of living systems. Single-stranded RNA (ssRNA), one of the possible genetic information carriers, is used as a genomic molecule in some viruses and experimental models of primitive life-forms in the hypothetical RNA or RNA–protein world (Higgs and Lehman 2015; Joyce and Szostak 2018). A possible method to overcome this problem is to use ssRNA that is likely to form intramolecular base-pairings (i.e., secondary structures), which hinder intermolecular base-pairings (i.e., doublestrand formation). The RNA evolved to be more efficiently replicated by the replicase with less dsRNA formation In this experiment, the population size of ssRNA was sufficiently high (∼108), which presumably covers all possible point mutations. We found that the numbers of unpaired G and C bases were gradually decreased as mutations accumulated These results support the importance of the fewer unpaired GC rule for efficient ssRNA replication by Qβ replicase

RESULTS

DISCUSSION

MATERIALS AND METHODS