1. Field of the Invention
Molecular markers are conventionally used in the electrophoretic separation and/or identification of microbiological materials. During polyacrylamide gel electrophoresis (PAGE) under denaturing conditions, circular RNAs usually migrate more slowly than the corresponding linear molecules, therefore an accurate system must accomodate both types of molecules. This invention relates to a novel method for the preparation of circular RNAs useful as markers and to an electrophoretic system capable of allowing the precise sizing of both linear and circular RNA molecules.
2. Description of the Related Art
Circular RNAs have been shown to play an active role in a number of important biological processes such as the infection of a number of plants. For example, the potato spindle tuber viroid, a small autonomously replicating pathogenic RNA, was the first reported naturally-occurring circular RNA. Subsequently, other covalently circular subviral RNAs were reported, including the satellite RNA of tobacco ringspot virus, satTRSV RNA (Sogo and Schneider. 1982. Virology. vol. 117, pp. 401-415) and the genome of hepatitis delta virus (Wang et al. 1986. Nature. vol. 323, pp. 508-514).
Circular RNAs are also generated by synthesis and processing of cellular mRNAs and rRNAs. For example, following self-excision from pre-rRNA, the group I intron of Tetrahymena thermophila circulizes via attack of its 3'-terminal hydroxyl group on a phosphodiester linkage near the 5'-terminus (see, for example, Kruger et al. 1982. Cell. vol. 31, pp. 147-157). In addition, spliceosome-mediated maturation of eukaryotic mRNA as well as self-excision of group II introns releases a branched or "lariat" RNA in which the 5'-terminus of the intron is joined to the free 2'-OH of an adenylate residue .about.25 nt upstream from the 3' splice site (reviewed in Padgett et al. 1986. Annu. Rev. Biochem. vol. 55, 1119-1150; Ferat and Michel. 1995. Annu. Rev. Biochem. vol. 64, pp. 435-461). Splice site pairing across an exon can also result in exon circularization in vitro (Pasman et al. 1996. RNA. vol. 2, pp. 603-610). These circularized RNAs have been found to be more resistant to degradation than the corresponding linear forms (Puttaraju et al. 1993. Nucleic Acids Res. vol. 21, pp. 4253-4258).
Production of circular RNAs has been attempted by various methods; however, in general, the process is complicated and requires extensive purification. Wang and Kool (1994. Nucleic Acids Research. vol. 22, no. 12, pp. 2326-2333), for example, disclosed a nonenzymatic synthetic method for the production of circular RNA oligonucleotides utilizing preparative 20% denaturing PAGE for purification purposes. Beaudry and Perrault (1995. Nucleic Acids Research. vol. 23, no. 15, pp. 3064-3066) described a synthetic method which involved PCR amplification of a particular gene sequence of unit length. It was considered an improvement over conventional techniques because it required only one purification step.
A number of electrophoresis systems capable of resolving circular and linear RNA have been described, but preparation of the molecular standards necessary for calibrating the gels can be laborious (e.g., Maniatis et al. 1975. Biochemistry. vol. 14, pp. 3787-3794; Singh and Boucher. 1987. Phytopathology. vol. 77, pp. 1588-1591; Schumacher et al. 1983. Anal. Biochem. vol. 135, pp. 288-295; Schumacher et al. A986. J. Phytopathol. vol. 115, 332-343).
An improved method which simplifies the preparation of circular RNAs for use as markers in PAGE systems and for a simplified PAGE system which accurately separates and identifies circular and linear RNAs would be advantageous in studies requiring accurate sizing of naturally-occurring linear and circular pathogenic RNAs associated with both animal and plant diseases and the abnormal processing of cellular messenger RNAs.