Abstract
The packaging RNA (pRNA) found in phi29 bacteriophage is an essential component of a molecular motor that packages the phage's DNA genome. The pRNA forms higher-order multimers by intermolecular "kissing" interactions between identical molecules. The phi29 pRNA is a proven building block for nanotechnology and a model to explore the rare phenomenon of naturally occurring RNA self-association. Although the self-association properties of the phi29 pRNA have been extensively studied and this pRNA is used in nanotechnology, the characteristics of phylogenetically related pRNAs with divergent sequences are comparatively underexplored. These diverse pRNAs may lend new insight into both the rules governing RNA self-association and for RNA engineering. Therefore, we used a combination of biochemical and biophysical methods to resolve ambiguities in the proposed secondary structures of pRNAs from M2, GA1, SF5, and B103 phage, and to discover that different naturally occurring pRNAs form multimers of different stoichiometry and thermostability. Indeed, the M2 pRNA formed multimers that were particularly thermostable and may be more useful than phi29 pRNA for many applications. To determine if diverse pRNA behaviors are conferred by different kissing loop sequences, we designed and tested chimeric RNAs based on our revised secondary structural models. We found that although the kissing loops are essential for self-association, the critical determinant of multimer stability and stoichiometry is likely the diverse three-way junctions found in these RNAs. Using known features of RNA three-way junctions and solved structures of phi29 pRNA's junction, we propose a model for how different junctions affect self-association.