RRC ID 49551
Author Yumura M, Yamamoto N, Yokoyama K, Mori H, Yomo T, Ichihashi N.
Title Combinatorial selection for replicable RNA by Qβ replicase while maintaining encoded gene function.
Journal PLoS One
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.
Volume 12(3)
Pages e0174130
Published 2017-3-22
DOI 10.1371/journal.pone.0174130
PII PONE-D-16-30302
PMID 28328998
PMC PMC5362092
MeSH Amino Acid Sequence / genetics Amino Acids / genetics Escherichia coli / genetics Escherichia coli / metabolism Phenotype Protein Domains / genetics Q beta Replicase / metabolism* RNA / genetics* RNA-Dependent RNA Polymerase / metabolism* beta-Galactosidase / genetics
IF 2.74
Times Cited 5
Resource
DNA material pUC-mdv-alphal180-mutL2 (RDB15410)