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Title: SYNTHESIS AND TWO-DIMENSIONAL ELECTROPHORETIC ANALYSIS OF MIXED POPULATIONSOF CIRCULAR AND LINEAR RNAS

Author
item FELDSTEIN, PAUL - UMCP, PLANT BIOLOGY DEPT.
item LEVY, LAURENE - USDA, APHIS
item RANDLES, JOHN - UNIV. OF ADELAIDE, S. AUS
item Owens, Robert

Submitted to: Nucleic Acids Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/10/1997
Publication Date: N/A
Citation: N/A

Interpretive Summary: Circular RNAs (including plant viroids and other subviral RNAs) are known to play an important role in many biological processes, but identification and characterization of such molecules is often hindered by the lack of a suitable set of standards. This manuscript describes (i) the construction of a series of modified tobacco ringspot virus satellite RNAs that are able to spontaneously self-circularize in vitro and (ii) a novel two-dimensional gel system capable of resolving mixtures of circular and linear RNAs containing 225-1132 nucleotides. Identification of a previously undescribed heterodimeric form of coconut cadang-cadang viroid among the RNA molecules extracted from infected palms illustrates the potential power of such an experimental strategy. These results will be of greatest interest to plant and animal virologists, especially those interested in the detection and characterization of new viral and subviral pathogens. Other interested parties might include small- to medium-sized biotechnology companies for commercializing this methodology.

Technical Abstract: Unlike the cleavage reactions carried out by many other self-cleaving RNAs, spontaneous cleavage of the less abundant form of tobacco ringspot virus satellite RNA is readily reversible. As shown by Feldstein and Bruening [Nucleic Acids, Res. 21, 1991-1998 (1993], small "minimonomer" RNAs derived from this molecule and containing little more than covalently attached ribozyme and substrate cleavage products are also able to efficiently circularize. We have capitalized on the spontaneous nature of this reaction to produce a series of self-circularizing RNAs. Mixtures of linear and circular RNAs synthesized in vitro and containing 225-1132 nucleotides could be completely resolved using a novel two-dimensional polyacrylamide gel electrophoresis system under denaturing conditions. Analysis of a complex mixture of coconut cadang-cadang viroid RNAs isolated from infected palms using this gel system revealed the presence of relatively large amounts of a previously undescribed "fast-slow" heterodimeric viroid RNA.