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

Usui, Kimihito; Ichihashi, Norikazu; Yomo, Tetsuya

To render this RNA replicable as a single strand, we first introduced 75 synonymous mutations that reduced the GC number in loops to <5 to obtain mutant m1. The predicted secondary structure of m1 exhibited reductions in size and the number of loops compared to the original ( Figure 7A , m1). The replication experiment with m1 indicated that the synthesis of ssRNA increased significantly, but the dsRNA ratio was still greater than 0.5 ( Figure 7B, m1) , likely because the mutant m1 still had six remaining loops that contained three or four GCs (Loops 1-6 of m1, Figure 7A ). Further reduction of the GC number by introducing synonymous mutations would be difficult in most of these loops because it would require massive reconstruction around the loop regions. Therefore, we first attempted to identify target loops that must be improved and then to reconstitute the structures around the loops. To identify the target loops, we deleted the remaining Loops 1-6 with various combinations to obtain the mutants m2-m12 and measured the dsRNA ratios of the replication products ( Table 1 ). The dsRNA ratios of these deletion mutants were all lower than that of m1. Among the deletion mutants, m3, which lost Loops 1-5, showed the lowest dsRNA ratio (0.34), and m11, which lost three loops (Loops 1, 2 and 4), lost the fewest loops, yet exhibited a dsRNA ratio of <0.5. We then attempted to modify the Loops 1, 2 and 4 regions to reduce the GC number by introducing synonymous mutations instead of deletions. For Loops 2 and 4, we reconstructed structures around the loops by introducing 10 synonymous mutations to decrease the GC number in the loops to <3. For Loop 1, which locates in the 5 -untranslated region, we simply deleted G and C in this loop to obtain mutant m13 or substituted the sequence of the loop with all U bases to obtain mutant m14. Both mutants (m13 and m14) exhibited dsRNA ratios of <0.5 (0.47 and 0.45, respectively, Figure 7B ) while maintaining the original amino acid sequence of the encoding gene.