In vivo decay kinetic parameters of hammerhead ribozymes.
Ribozymes offer a potentially important way to inactivate intracellular RNA from almost any gene whose nucleotide sequence is known. Recently, 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 different 3'-terminal hairpins on ribozyme stability as well as their effect 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 from a transcription terminator of bacteriophage T7, another contained the same hairpin but modified to be highly enriched for G+C nucleotides, and a third lacked a hairpin. The TNF alpha ribozyme decay had two kinetic components. The slow component exhibited exponential decay with a half life of approximately 250 h in all cases. The 3'-terminal hairpin has no significant effect on this component. This slow phase accounted for 60-80% of ribozyme decay. The rapid phase also exhibited exponential decay. For this phase, a 3'-terminal hairpin roughly doubled the half-life (1.7-3.4). The slow phase of degradation was about three times faster for a ribozyme directed at the integrase mRNA of human immunodeficiency virus-1 than that seen with the TNF alpha ribozyme. Taken together, these results suggest that the ribozyme population is initially sensitive to degradation, with the presence of a hairpin provides some protection, and indicate that the addition of the hairpin to the ribozyme did not prevent the in vivo additional stabilizing effect of the protein(s).
Sioud
M
M
Institute of Immunology and Rheumatology, Rikshospitalet, Oslo, Norway.
Opstad
A
A
Zhao
J Q
JQ
Levitz
R
R
Benham
C
C
Drlica
K
K
D001483:Base Sequence; D002460:Cell Line; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D016337:RNA, Catalytic / Q000737:chemistry*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D016337:RNA, Catalytic; D014409:Tumor Necrosis Factor-alpha