Detection and analysis of hairpin II, an essential metastable structural element in viroid replication intermediates

In (-)-stranded replication intermediates of the potato spindle tuber viroid (PSTVd) a thermodynamically metastable structure containing a specific hairpin structure (HP II) has been proposed to be essential for viroid replication. In the present work a method was devised allowing the direct detection of the HP II structure in vitro and in vivo using a biophysical approach. An RNA oligonucleotide was constructed which specifically binds to the HP II loop region in transient (-)-strand intermediates. Analysis of the resulting oligonucleotide/HP II complexes on temperature-gradient gels enabled us to follow the formation of HP II during in vitro transcription by T7 RNA polymerase. Moreover, we were able to demonstrate the formation of HP II during viroid replication in potato (Solanum tuberosum) cells.