A method for generating transcripts with defined 5″ and ′3 termini by autolytic processing

Ribozyme mediated cleavage of acute phase serum amyloid A (A‐SAA) mRNA in vitro

“…This increase in size may, however, prevent access of the ribozyme to the target site. cis-Cleaving autocatalytic ribozymes have been previously used to generate RNA transcripts with defined 5'-and 3'-termini [21,22]. We have therefore designed an autocatalytic cassette (RpSAA3') that contains RzSAA immediately upstream of another ribozyme and is capable of folding back and forming a hammerhead ribozyme using the 3' end of RzSAA as the target substrate and cleaving between the two (Fig.…”

A Triplex Ribozyme Expression System Based on a Single Hairpin Ribozyme

“…Early triplex systems based on three hammerhead RZs units resulted in the ef cient in vivo inhibition of HIV-1 (Yuyama et al, 1994) and down-regulation of the retinoblastoma gene (Benedict et al, 1998). Nevertheless, the complex engineering of multiplex triplex constructs based in triple-RZ units may result in recombination due to the sequence similarities between the multiple cis-cleaving and trans-cleaving RZs (Altschuler et al, 1992).…”

High-performance liquid chromatography purification of homogenous-length RNA produced by trans cleavage with a hammerhead ribozyme

“…Various cis and trans ribozyme systems have been described for eliminating heterogeneity at the 39 and 59-termini of in vitro-transcribed RNAs (Dzianott & Bujarski, 1988;Grosshans & Cech, 1991;Altschuler et al+, 1992;Price et al+, 1995;Ferré D'Amaré & Doudna, 1996)+ For cleavage in cis, the RNA is usually transcribed from a plasmid or PCR DNA template that contains a cis-cleaving ribozyme at the 39-terminus of the RNA (and also at the 59-terminus if desired)+ This procedure is better suited for longer RNAs, because both the transcription and cis cleavage reactions are taking place simultaneously+ If the transcription produces abortive RNAs similar in length to the desired product then it is not possible to separate these aborts from the cleaved product RNA utilizing the cis-cleavage procedure (see the gel of the transcription reaction shown in…”

“…Various RNA-substrate trans-cleaving hammerhead systems studied in this work+ For each substrate RNA, the nucleotides designed to base pair with the processing hammerhead are in outline font, and intermolecular base pairs are designated by solid lines, intramolecular (hammerhead-substrate) base pairs are designated by dashed lines, and the site of cleavage is indicated by the arrow+ The trans-cleaving hammerhead for each RNA is shown in italic and the Nϩ1 nucleotides at the 39 end of the RNA substrates are designated by X+ In A, the 11 conserved nucleotides required for hammerhead activity are boxed, the nucleotide at the cleavage site (which can be any base but G) is circled and stems 1-3 in the hammerhead ribozyme-substrate complex are denoted+ A: a bFGF-binding RNA; B: a theophylline-binding RNA; C: a faster-cleaving hammerhead ribozyme variant; and D: an RNA that contains specific metal binding sites+ while also eliminating the most laborious step in largescale RNA preparation, the purification of RNA transcripts by denaturing PAGE+ The procedure presented here will be compared with other methods for eliminating 39 heterogeneity in RNAs that are generated by in vitro transcription (Dzianott & Bujarski, 1988;Grosshans & Cech, 1991;Altschuler et al+, 1992;Price et al+, 1995;Ferré D'Amaré & Doudna, 1996;Lapham & Crothers, 1996;Doudna, 1997)+…”