A design principle for a single-stranded RNA genome that replicates with less double-strand formation

Usui, Kimihito; Ichihashi, Norikazu; Yomo, Tetsuya

gene through in vitro translation and the ␣ complementation assay. The RNAs (mdv-␣, m1, m3, m13 and m14) were mixed with a reconstituted translation system containing a fluorescent substrate and the omega protein, and the fluorescence was monitored during the incubation ( Figure 7D ). For mutant m3, which lost Loops 1-5, the fluorescence was at the same level as that with no RNA, while for the original mdv-␣, mutants m14, m1 and m13 showed higher fluorescence than that with no RNA, indicating that these RNAs, except for m3, encoded active ␣-domain genes. Taken together, these results demonstrate that based on the design rule, we succeeded in developing RNAs (m13 and m14) that possess the capacity to replicate continuously as a single strand and encode an active gene.