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

Usui, Kimihito; Ichihashi, Norikazu; Yomo, Tetsuya

To date, the dsRNA formations that are produced during replication have been extensively investigated using Q␤ replicase, an RNA-dependent RNA polymerase from bacteriophage Q␤, which synthesizes a complementary strand in a primer-independent manner (6) . The molecular mechanism of the replication has been intensively studied recently (7) (8) (9) . This replicase requires CCC at the 3 -teminus (10) , stem structures at the 5 -terminus (11) , an internal C/Urich domain (12) and a long-range interaction between 5and 3 -termini (13) for efficient replication. However, even if all of these requirements are satisfied, RNA-encoding genes that originate from organisms other than bacteriophage Q␤ mainly produce dsRNA and then terminate replication (14) (15) (16) (17) (18) (19) (20) (21) . Although some factors, such as components in a cell-free translation system and ribosomal protein S1, are known to inhibit dsRNA formation (22) (23) (24) , they were insufficient to render ssRNA-encoding arbitrary genes replicable without dsRNA formation (22) . In addition, rigid secondary structures of a template RNA have been reported to inhibit dsRNA formation (25, 26) . Because quantitative requirements for the structures (i.e. the size or stability of structures) have not been determined, it is not possible to design an ssRNA that encodes an arbitrary gene at this time.