Design and Function of Triplex Hairpin Ribozymes

Abstract

Triplex ribozymes allow for the individual activity of multiple trans-acting ribozymes producing higher target cleavage relative to tandem-expressed RZs. A triplex expression system based on a single hairpin ribozyme for the multiple expression (multiplex) vectors can be engineered to target RNAs with single or multiple antisense-accessible sites. System construction relies on triplex expression modules consisting of hairpin ribozyme cassettes flanked by ribozymes lacking catalytic domains. Multiplex vectors can be generated with single or multiple specificity by tandem cloning of triplex expression modules. Triplex ribozymes are initially tested in vitro using cis- and trans-cleavage assays against radioactive-labeled targets. In addition, triplex ribozymes are tested for cis and trans cleavage in vivo by transfection in cultured cells followed by ribonuclease protection assays (RPAs) and RT-PCR. The use of triplex configurations with multiplex ribozymes will provide the basis for the development of future RZ-based therapies and technologies.

Therapeutic startegies for human papillomavirus infection and associated cancers

“…Ribozyme targeting HPV16 E6E7 transcripts (Rz170) suppressed cell growth and sensitized cervical cancer cells to chemotherapy and radiotherapy (120). To counteract size related problems due to cloning in plasmid vectors, the construct was modified to triplex ribozyme expression system that released processed ribozymes as a single transcript by a self-processing mechanism (121,122). However, these modifications did not increase ribozyme mediated inhibition more than 30% (122), and thus this approach required further improvement to make ribozymes a therapeutic moiety.…”

“…To counteract size related problems due to cloning in plasmid vectors, the construct was modified to triplex ribozyme expression system that released processed ribozymes as a single transcript by a self-processing mechanism (121,122). However, these modifications did not increase ribozyme mediated inhibition more than 30% (122), and thus this approach required further improvement to make ribozymes a therapeutic moiety. Further, the catalytic activity the ribozymes found to dramatically drop within the cellular environment due to Mg ++ availability, nuclease action protein binding and with reduced probability of co-localization with the target.…”