Rapid evolution of in vivo-selected sequences and structures replacing 20% of a subviral RNA.

The 356nt noncoding satellite RNA C (satC) of Turnip crinkle virus (TCV) is composed of 5′ sequences from a second TCV satRNA (satD) and 3′ sequences derived from TCV. SHAPE structure mapping revealed that 76nt in the poorly-characterized satD-derived region form an extended hairpin (H2). Pools of satC in which H2 was replaced with 76, 38, or 19 random nt were co-inoculated with TCV helper virus onto plants and satC fitness assessed using in vivo functional selection (SELEX). The most functional progeny satCs, including one as fit as wild-type, contained a 38–39nt H2 region that adopted a hairpin structure and exhibited an increased ratio of dimeric to monomeric molecules. Some progeny of satC with H2 deleted featured a duplication of 38nt, partially rebuilding the deletion. Therefore, H2 can be replaced by a 38–39nt hairpin, sufficient for overall structural stability of the 5′ end of satC.