Discrimination of a single base change in a ribozyme using the gene for dihydrofolate reductase as a selective marker in  Escherichia coli

Abstract

For use of ribozymes in vivo, it is desirable to select functional ribozymes in the cellular environment (in the presence of inhibitory factors and limited concentrations of mandatory Mg 2؉ ions, etc.). As a first step toward this goal, we developed a new screening system for detection in vivo of an active ribozyme from pools of active and inactive ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker. In our DHFR expression vector, the sequence encoding either the active or the inactive ribozyme was connected to the DHFR gene. The plasmid was designed such that, when the ribozyme was active, the rate of production of DHFR was high enough to endow resistance to trimethoprim (TMP). We demonstrated that the active ribozyme did indeed cleave the primary transcript in vivo, whereas the inactive ribozyme had no cleavage activity. Cells that harbored the active-ribozyme-coding plasmid grew faster in the presence of a fixed concentration of TMP than the corresponding cells that harbored the inactive-ribozyme-coding plasmid. Consequently, when cells were transformed by a mixture that consisted of active-and inactive-ribozyme-coding plasmids at a ratio of 1:1, (i) mainly those cells that harbored active ribozymes survived in the presence of TMP and (ii) both active-and inactive-ribozyme-harboring cells grew at an identical rate in the absence of TMP, a demonstration of a positive selection system in vivo. If the background ''noise'' can be removed completely in the future, the selection system might usefully complement existing selection systems in vitro.

Analysis of inhibitory action of modified U1 snRNAs on target gene expression: discrimination of two RNA targets differing by a 1 bp mismatch

“…Antisense oligonucleotides and RNAs are ineffective for this degree of discrimination (6,7). Ribozymes have the potential to discriminate a 1 bp mismatch, however, varying levels of inhibition of the mutation-bearing transcript have been reported (8)(9)(10)(11). Modified U1 snRNA might be able to complement a ribozyme in achieving allele-specific RNA suppression because it works by a fundamentally different mechanism and may have the potential to discriminate two transcripts that differ by 1-2 bp.…”

Factors Governing the Activity In Vivo of Ribozymes Transcribed by RNA Polymerase III

“…Because of its small size and potential as an antiviral agent, numerous mechanistic studies (10,11,13,31,37,(57)(58)(59)(60) and studies directed towards application in vivo have been performed (13,14,32,38,40,42,50). Many successful experiments aimed at the use of ribozymes for suppression of gene expression in different organisms have been reported (12,15,16,24,27,30,41,53,55,56). However, the efficacy of ribozymes in vitro is not necessarily correlated with functional activity in vivo.…”

RNA hairpin loops repress protein synthesis more strongly than hammerhead ribozymes

“…Although many studies have been carried out, which purport to show reduction of gene activity by a ribozyme in bacterial, mammalian or plant cell models, there remains no obvious path for the application of ribozymes to human medicine or agriculture except in a few cases [27,49,50]. Here, existing ribozyme activities appear too weak to be generally useful, and remain almost undetectable when studying full-length mRNA in vivo.…”