Synthesis and two-dimensional electrophoretic analysis of mixed population of circular and linear RNAs:

Spontaneous cleavage of the less abundant form tobacco ringspot virus satellite RNA is readily reversible. Capitalizing on earlier observations by Feldstein and Bruening that small 'mini-monomer' RNAs derived from this molecule and containing little more than covalently attached ribozyme and substrate cleavage products are able to efficiently circularize, we have constructed a series of self-circularizing RNAs of precisely, known size. Mixtures of linear and circular RNAs synthesized in vitro and containing 225-1132 nt could be completely resolved using a novel two-dimensional denaturing polyacrylamide gel electrophoresis system. Similar analyses of a complex mixture of coconut cadang-cadang viroid RNAs revealed the presence of relatively large amounts of a previously undescribed 'fast-slow' heterodimeric RNA species in infected palms. Only a single DNA template is required to prepare each pair of circular and linear RNA markers.

image caption - Analysis of the complex mixture of monomeric and dimeric viroid RNAs present in CCCVd-infected palms. Unlabeled CCCVd RNAs were fractionated by two-dimensional electrophoreses and transferred to a positively charged nylon membrane before hybridization with a ³²P-labeled RNA probe complementary to CCCVd. In addition to circular and linear forms of the monomeric fast(CCCVd_F, 246-247 nt) and slow (CCCVd_S, 296-297nt) forms of CCCVd RNAs, three different dimeric RNAs could also be resolved, i.e. fast-fast(CCCVd_FF), fast-slow(CCCVd_FS) and slow-slow (CCCVd_SS) molecules.