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.
Department of Surgery, University of Florida College of Medicine, 1600 S.W. Archer Road, Gainesville, Florida, 32610, USA. smackay@surgery.ufl.edu
S L
SL
MacKay
C L
CL
Tannahill
T
T
Auffenberg
R
R
Ksontini
E M
EM
Copeland
L L
LL
Moldawer
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