Combinatorial selection for replicable RNA by Qβ replicase while maintaining encoded gene function.
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.
Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka, Japan.
Mio
M
Yumura
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama, Ikoma, Japan.|Department of Biomedical Ethics and Public Policy, Graduate School of Medicine, Osaka University, 2-2, Yamadaoka, Suita, Osaka, Japan.
Natsuko
N
Yamamoto
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama, Ikoma, Japan.
Katsushi
K
Yokoyama
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama, Ikoma, Japan.
Hirotada
H
Mori
East China Normal University, Shanghai, P.R.China.
Tetsuya
T
Yomo
Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka, Japan.|Graduate School of Frontier Biosciences, Osaka University 1-5 Yamadaoka, Suita, Osaka, Japan.
Norikazu
http://orcid.org/0000-0001-7087-2718
N
Ichihashi
D000595:Amino Acid Sequence / Q000235:genetics; D000596:Amino Acids / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D000072417:Protein Domains / Q000235:genetics; D011777:Q beta Replicase / Q000378:metabolism*; D012313:RNA / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics
D000596:Amino Acids; D012313:RNA; D011777:Q beta Replicase; D012324:RNA-Dependent RNA Polymerase; D001616:beta-Galactosidase