The elucidation of the structures of viroids, noncoding infectious RNA species, is paramount to obtain an understanding of the various aspects of their life cycles (including replication, transport and pathogenesis). In general, the secondary structures of viroids have been predicted using computer software programs which have been shown to possess several important limitations. Clearly, the predicted structure of a viroid needs to receive physical support prior to its use in the accurate interpretation of any mechanistic studies. Here, SHAPE probing coupled to computer-assisted structure prediction using the RNAstructure software program was employed to determine the structures of five viroids. These species belong to four genera of the Pospiviroidae family, and none have had their structure characterized in solution. In addition, several interesting questions were addressed by either studying various sequence variants or varying the SHAPE conditions. More importantly, this approach is novel in the study of viroids, and should be of significant aid in the determination of the structures of other RNA species.
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