DNAzyme-mediated silencing of ornithine decarboxylase.
The value of reducing the activity of ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is well-appreciated. Polyamines are necessary components for cell growth, and manipulation of polyamine homeostasis may be an effective strategy for the treatment of a number of disorders, including neoplastic diseases. An approach to develop an effective DNAzyme, using the 10-23 model, against ODC is described in these studies. DNAzymes able to cleave the target ODC RNA were identified in vitro and further characterized by the effect each had on ODC protein and activity levels using in vitro translated ODC RNA. ODC protein levels and activity correlated well with the RNA cleavage activity of the DNAzyme. One of the DNAzymes, DZ IV, which exhibited good activity, was optimized for use in cell culture studies. The DNAzyme hybridization arms were altered from equal length arms varying in length (8, 9, 10, or 11 nucleotides) or to unequal length arms (7/11 nucleotides), and kinetic analyses were performed to identify the most catalytically efficient configuration. DZ IV with equal arms nine nucleotides in length proved to be the most catalytically efficient. In HEK 293 cells, DZ IV was able to reduce the amount of translated ODC protein, resulting in approximately 80% reduction in ODC activity-a statistically significant enhancement over the apparent antisense effect of a catalytically inactive DNAzyme. These results indicate that this DNAzyme may be a useful tool to study the function of ODC and may have potential therapeutic uses.
Ackermann
Joseph M
JM
Department of Pharmacology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
Kanugula
Sreenivas
S
Pegg
Anthony E
AE
D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D021881:DNA, Catalytic / Q000138:chemical synthesis / Q000737:chemistry*; D004789:Enzyme Activation / Q000235:genetics; D020868:Gene Silencing; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D051379:Mice; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D009955:Ornithine Decarboxylase / Q000235:genetics* / Q000378:metabolism*; D065108:Ornithine Decarboxylase Inhibitors; D010084:Oxidation-Reduction; D014176:Protein Biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000138:chemical synthesis / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics; D014162:Transfection
D021881:DNA, Catalytic; D009838:Oligodeoxyribonucleotides; D065108:Ornithine Decarboxylase Inhibitors; D012333:RNA, Messenger; D009955:Ornithine Decarboxylase