Generation of RNA ladders by rolling circle transcription of small circular oligodeoxyribonucleotides.

As experimental methods in molecular biology have rapidly progressed, the need for simple methodology to create RNA size markers has become increasingly apparent. Ribosomal RNAs are commonly used as size markers for larger RNAs in denaturing agarose gels (9); however, RNA markers are not commercially available for the characterization of <100 nucleotide (nt) RNAs by electrophoresis. This fact, as well as the inherent instability of RNA vs. DNA, prompted the use of DNA size markers in early research. However, denatured DNA strands migrate faster than RNA, introducing a potential source of error in RNA size determination (11,14). For these reasons, several research groups have recently worked to find improved methods for generating RNA size markers of varied lengths. RNA size estimation has been carried out by visualization of three or four mRNAs of known length using Northern blotting techniques (12) or by using the products of transcription of several DNA templates containing the appropriate promoter sequence using a radioactive nucleotide (8,10). These methods are relatively labor intensive and require considerable manipulation to generate new length markers. A more recent report used a single plasmid, linearized by 13 readily available restriction enzymes to produce 13 DNA templates (1). Transcription provided three sets of RNA markers (148–3621 nt) corresponding to low, medium and high size ranges that provide for significant flexibility in application. However, none of these approaches offers either markers for small RNAs (<100 nt) or a means for generating regularly spaced ladders. A different method for production of RNA size markers has made use of the inherent cleavage activity of tandem, cis-cleaving hammerhead and hairpin ribozymes to segment a large RNA transcript (2). Transcription of a plasmid that encodes these ribozymes produces RNA that, after ribozyme cleavage, contains discrete RNAs ranging from 83–244 nt. This approach uses only a single plasmid to create six fragments; however, the size range of RNAs produced from this system is relatively small, and ribozyme size variation was not explored. The above work has made RNA markers more accessible than they once were; however, there remains a need for developing even simpler, more generally useful methods. RNA ladders with regular band spacing would be particularly useful because they would facilitate more accurate size estimation by giving a greater number of markers, thus allowing for better interpolation between markers. Our approach to the synthesis of RNA size markers, like those of previous workers (2), uses ribozyme activity to create a ladder of RNA size markers. However, the method we use to synthesize the RNAs is, to our knowledge, unique. While previous approaches have involved the synthesis of RNA transcripts from a linearized plasmid DNA template, our ribozyme RNAs are produced by rolling circle transcription (RCT) of single-stranded DNA (ssDNA) circles (Figure 1). We have previously shown that RCT can be used to generate both hammerhead (3) and hairpin (5) ribozymes. We now demonstrate that regularly spaced RNA ladders can be obtained by transcription of DNA circles of various sizes (63–83 nt) encoding these ribozyme motifs. To demonstrate a range of marker sizes, we constructed four differently sized circular ssDNAs, altering domains not required for ribozyme activity. Two of these DNA circles encode hairpin ribozymes (67TRSV and 73TRSV), and two encode hammerhead ribozymes (63ASBV and 83ASBV). These sequences are subsets (or close variants) of known viroids (15) and virusoids (6). A simple “one-pot” cyclization scheme using T4 DNA ligase and short synthetic DNA splints is used for preparation of ssDNA circles (4). Briefly, each ligation proceeds from two short linear DNAs (precursor segments) that are intermolecularly joined to give a linear full-length pre-circle using a templating oligonu-