IN-VIVO DECAY KINETIC-PARAMETERS OF HAMMERHEAD RIBOZYMES

Ribozymes offer a potentially important way to inactivate intracellula r RNA from almost any gene whose nucleotide sequence is known. Recentl y, we found that hammerhead ribozymes directed against mRNA of tumour necrosis factor alpha (TNF alpha) and its derivatives, preferentially bind to a cellular protein(s). To better understand the effect of diff erent 3'-terminal hairpins on ribozyme stability as well as their effe ct on the protein binding to the ribozyme, a mathematical treatment of the decay of three TNF alpha ribozymes that differed at their 3' ends was performed. One ribozyme contained a 3'-terminal hairpin derived f rom a transcription terminator of bacteriophage T7, another contained the same hairpin but modified to be highly enriched for G+C nucleotide s, and a third lacked a hairpin. The TNF alpha ribozyme decay had two kinetic components. The slow component exhibited exponential decay wit h a half life of approximately 250 h in all cases. The 3'-terminal hai rpin has no significant effect on this component. This slow phase acco unted for 60 - 80% of ribozyme decay. The rapid phase also exhibited e xponential decay. For this phase, a 3'-terminal hairpin roughly double d the half-life (1.7 - 3.4). The slow phase of degradation was about t hree times faster for a ribozyme directed at the integrase mRNA of hum an immunodeficiency virus-1 than that seen with the TNF alpha ribozyme . Taken together, these results suggest that the ribozyme population i s initially sensitive to degradation, with the presence of a hairpin p rovides some protection, and indicate that the addition of the hairpin to the ribozyme did not prevent the in vivo additional stabilizing ef fect of the protein(s).