Combinatorial selection for replicable RNA by Qβ replicase while maintaining encoded gene function
The selection cycle consists of two types of selection: in vitro selection of more replicable RNA and in vivo functional selection of the α-domain gene of β-galactosidase. (1) DNA fragments containing the α-domain gene, the terminal recognition sites for Qβ replicase, and the T7 promoter were amplified, and mutations were introduced by error-prone PCR to obtain a DNA library. (2) The DNA library was transcribed in vitro with T7 RNA polymerase to produce the RNA library. (3) In vitro RNA replication was performed with Qβ replicase. In this step, more replicable mutant RNAs in the library were selected. (4) The replicated RNAs were reverse transcribed into cDNAs, (5) ligated to a plasmid vector and (6) transformed into an E. coli strain that expressed the ω-domain of β-galactosidase. (7) The transformed E. coli were plated on an agar plate containing the colorimetric substrate of β-galactosidase. The cells harboring the plasmids containing the functional α-domain gene produced blue-colored colonies and were collected. (8) The plasmids were then extracted from the colony mixture for the next cycle.