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

Usui, Kimihito; Ichihashi, Norikazu; Yomo, Tetsuya

Although we found a simple rule for an RNA that replicates with less dsRNA formation, it is not guaranteed that we can develop a continuously replicable RNA by following this rule. More importantly, there is also no guarantee that we can find a sequence that both satisfies the rule and maintains the activity of the encoded gene. To examine the usefulness of this rule, we attempted to design an ssRNA that is con-tinuously replicable as a single strand and that encodes an active gene by following this rule. As a criterion of 'continuously replicable as a single strand,' we employed the condition of 'less than 0.5 dsRNA ratio,' because if the dsRNA ratio becomes >0.5, the ssRNA only decreases as replication proceeds. As a target gene, we used the ␣-domain of ␤galactosidase of Escherichia coli. The activity of this small gene (∼300 nt) is monitored by ␣ complementation in the presence of the omega-domain of ␤-galactosidase (31, 37) . We introduced the ␣-domain into the same site of MDV-1 as with the RNAs in Figure 2 to obtain mdv-␣ RNA (original mdv-␣). Secondary structure prediction of this RNA revealed the existence of many large loops ( Figure 7A , original mdv-␣). We performed the replication experiment with this RNA and found that almost all synthesized RNA was dsRNA ( Figure 7B, original) .