Combinatorial selection for replicable RNA by Qβ replicase while maintaining encoded gene function.

Mio Yumura,Tetsuya Yomo,Norikazu Ichihashi,Hirotada Mori,Natsuko Yamamoto,Katsushi Yokoyama,Sukesh R Bhaumik

doi:10.1371/journal.pone.0174130

Mio Yumura, Tetsuya Yomo + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0174130

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Abstract

Construction of a complex artificial self-replication system is challenging in the field of in vitro synthetic biology. Recently, we developed a translation-coupled RNA replication system, wherein an artificial genomic RNA replicates with the Qβ RNA replicase gene encoded on itself. The challenge is to introduce additional genes into the RNA to develop a complex system that mimics natural living systems. However, most RNA sequence encoding genes are not replicable by the Qβ replicase owing to its requirement for strong secondary structures throughout the RNA sequence that are absent in most genes. In this study, we establish a new combinatorial selection method to find an RNA sequence with secondary structures and functional amino acid sequences of the encoded gene. We selected RNA sequences based on their in vitro replication and in vivo gene functions. First, we used the α-domain gene of β-galactosidase as a model-encoding gene, with functional selection based on blue-white screening. Through the combinatorial selection, we developed more replicable RNAs while maintaining the function of the encoded α-domain. The selected sequence improved the affinity between the minus strand RNA and Qβ replicase. Second, we established an in vivo selection method applicable to a broader range of genes by using an Escherichia coli strain with one of the essential genes complemented with a plasmid. We performed the combinatorial selection using an RNA encoding serS and obtained more replicable RNA encoding functional serS gene. These results suggest that combinatorial selection methods are useful for the development of RNA sequences replicable by Qβ replicase while maintaining the encoded gene function.

Highlights

In the field of in vitro synthetic biology or artificial cell synthesis, in vitro synthesis of several types of biological functions is being attempted to understand the design principles of biological systems or to develop new technologies [1,2,3,4,5,6,7]
We found that introduction of a new gene into the RNA genome completely abolished replication by the Qβ replicase because the replicase requires strong secondary structures throughout the RNA, which are absent in most genes [20]
Kinetic analysis of one clone (m4) revealed that the affinity between its minus strand and Qβ replicase was improved attributed to at least two point mutations, A87C and G116U. We developed another type of combinatorial cycle applicable to a broader range of genes and obtained more replicable RNA encoding functional serS gene

Summary

Introduction

In the field of in vitro synthetic biology or artificial cell synthesis, in vitro synthesis of several types of biological functions is being attempted to understand the design principles of biological systems or to develop new technologies [1,2,3,4,5,6,7]. We attempted this strategy and succeeded in developing an RNA sequence encoding the α-domain gene of β-galactosidase to be replicable at a certain level [20]. This strategy relies on RNA structure prediction algorithms, which are only accurate for short sequences and require several rounds of trial-and-error. We needed another strategy that is applicable to larger RNAs. In this study, we attempted to establish an evolutionary strategy to obtain a replicable RNA while maintaining the encoded gene function. We repeated this cycle for 10 rounds, and obtained more replicable RNAs that encode the functional genes

Result

Discussion

Materials and methods