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

Usui, Kimihito; Ichihashi, Norikazu; Yomo, Tetsuya

According to the model, the dsRNA ratio should also depend on the stability of the partial dsRNAs that are formed during replication, and the stability should depend on the GC ratio in the hybridized region. Hence, the GC ratio in the inserted loop sequence is expected to affect the dsRNA ratio. To examine this prediction, we performed replication experiments using RNAs with insert sequences that consist of only U and A (6. Loop UA5, 7. Loop UA10, 8. LoopUA15 and 9. Loop UA20) and compared to the results with the loops that consist of half G and half A described above. In contrast to the G and A loops, the dsRNA ratio of the RNA containing U and A loops did not increase, even with the insertion of a 20-nt loop ( Figure 3D ). To further investigate the effect of the GC ratio, we compared the dsRNA ratios of the RNAs with 10-nt loops inserted with various nucleotide compositions, such as all G (10. Loop G10), all A (11. Loop A10), all U (12. Loop U10), G and A (4. Loop GA10) and U and A (7. Loop UA10). The dsRNA ratio exhibited larger values for two RNAs (Loop G10 and GA10) that had higher GC ratios (100% and 50%, respectively, Figure 3F ). These results support the notion that the GC ratio, and hence the stability of the partial dsRNA formation between loops, is an important factor for dsRNA formation.