Combinatorial selection for replicable RNA by Qβ replicase while maintaining encoded gene function
A) Four RNA clones were randomly chosen after the selection cycle. These RNAs (0.1 nM) were replicated with the Qβ replicase (100 nM) for 1 h at 37°C and subjected to 8% polyacrylamide-gel electrophoresis followed by RNA staining with SYBR green II. SS and DS indicate the position of single- or double-strands, respectively. B) To obtain quantitative data, the RNA concentration of the original and m4 RNA in the replicated solutions were measured by quantitative PCR after reverse-transcription. Error bars represent standard error. C) The α-complementation activities of the α-domain genes. The plasmids encoding each RNA clone were introduced into an E. coli strain expressing the ω-domain gene, and the cells were streaked on a plate containing the colorimetric substrate for β-galactosidase. If the plasmids contain functional α-domain gene, the cell streaks become blue.