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

Usui, Kimihito; Ichihashi, Norikazu; Yomo, Tetsuya

To further investigate the effect of the stability of stem structures, we introduced 4-8 mismatch mutations into the 40-nt stem structure (20. Stem GAmis4, 21. Stem GAmis6 and 22. Stem GAmis8). The mismatch mutations did not affect the total RNA synthesis ( Figure 4E ) but significantly increased the dsRNA ratio when the mismatch number exceeded 4 ( Figure 4F ), indicating that the mismatch pair in stem structures should be at least less than 10% to inhibit dsRNA formation. In the experiments described above, we used RNAs that form primarily single stable structures, but generally, RNA possesses several semi-stable structures. We next investigated the effects of such multiple stable structures. We designed and inserted six sequences that form two different stable structures (one large stem or two small stems) with different ratios (23-28, transition stems 1-6). These sequences were designed to stabilize the two small stems rather than the one large stem as the number increased. The analysis of the replication products of these RNAs revealed that neither the total RNA synthesis nor the dsRNA ratios significantly differed among all of the RNAs ( Figure 5A and B), indicating that the existence of two different stable structures does not affect the dsRNA ratio.