Characterization in vitro and in vivo of hammerhead ribozymes directed against murine tumor necrosis factoralpha.
A hammerhead ribozyme directed against murine TNFalpha (mTNFalpha) mRNA has been constructed. In vitro studies showed that this ribozyme was released from the parent molecule by flanking cis-acting hammerhead and hairpin ribozymes. This same anti-mTNFalpha ribozyme specifically cleaved both synthetically derived substrate RNA and mTNFalpha mRNA within a pool of total cellular RNA. Endogenous delivery of this anti-mTNFalpha ribozyme via the self-cleaving cassette reduced mTNFalpha mRNA and protein levels in lipopolysaccharide (LPS)-stimulated, stably transfected murine macrophage RAW 264.7 cells. When complexed to liposomes and exogenously delivered to mouse peritoneal macrophages, the same ribozyme, with and without the cis-acting ribozymes, reduced mTNFalpha protein levels. However, an irrelevant ribozyme delivered in an identical fashion was also effective at reducing mTNFalpha protein levels. These data suggest that anti-mTNFalpha ribozymes can be constructed which efficiently cleave mTNFalpha mRNA, but irrelevant RNA/liposome complexes also effectively limit TNFalpha mRNA expression and can mimic functional ribozyme activity under in vitro conditions.
MacKay
S L
SL
Department of Surgery, University of Florida College of Medicine, 1600 S.W. Archer Road, Gainesville, Florida, 32610, USA. smackay@surgery.ufl.edu
Tannahill
C L
CL
Auffenberg
T
T
Ksontini
R
R
Copeland
E M
EM
Moldawer
L L
LL
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006868:Hydrolysis; D051379:Mice; D008810:Mice, Inbred C57BL; D016372:RNA, Antisense / Q000235:genetics; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D016372:RNA, Antisense; D016337:RNA, Catalytic; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases