SYNTHESIS AND 2-DIMENSIONAL ELECTROPHORETIC ANALYSIS OF MIXED POPULATIONS OF CIRCULAR AND LINEAR RNA

Spontaneous cleavage of the less abundant form of tobacco ringspot vir us satellite RNA is readily reversible. Capitalizing on earlier observ ations by Feldstein and Bruening that small 'mini-monomer' RNAs derive d from this molecule and containing little more than covalently attach ed ribozyme and substrate cleavage products are able to efficiently ci rcularize, 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 usin g a novel two-dimensional denaturing polyacrylamide gel electrophoresi s system. Similar analyses of a complex mixture of coconut cadang-cada ng viroid RNAs revealed the presence of relatively large amounts of a previously undescribed 'fast-slow' heterodimeric RNA species in infect ed palms. Only a single DNA template is required to prepare each pair of circular and linear RNA markers.