Characterization of a Potent and Specific Class of Antisense Oligonucleotide Inhibitor of Human Protein Kinase C-α Expression
0301 basic medicine
Protein Kinase C-alpha
Base Sequence
Dose-Response Relationship, Drug
Proto-Oncogene Proteins c-jun
Molecular Sequence Data
Thionucleotides
Gene Expression Regulation, Enzymologic
Oligodeoxyribonucleotides, Antisense
Isoenzymes
03 medical and health sciences
Tumor Cells, Cultured
Humans
Tetradecanoylphorbol Acetate
Thermodynamics
RNA, Messenger
Enzyme Inhibitors
Proto-Oncogene Proteins c-fos
Protein Kinase C
DOI:
10.1074/jbc.274.3.1715
Publication Date:
2002-07-26T14:56:47Z
AUTHORS (6)
ABSTRACT
The use of antisense oligonucleotides to inhibit the expression of targeted mRNA sequences is becoming increasingly commonplace. Although effective, the most widely used oligonucleotide modification (phosphorothioate) has some limitations. In previous studies we have described a 20-mer phosphorothioate oligodeoxynucleotide inhibitor of human protein kinase C-alpha expression. In an effort to identify improved antisense inhibitors of protein kinase C expression, a series of 2' modifications have been incorporated into the protein kinase C-alpha targeting oligonucleotide, and the effects on oligonucleotide biophysical characteristics and pharmacology evaluated. The incorporation of 2'-O-(2-methoxy)ethyl chemistry resulted in a number of significant improvements in oligonucleotide characteristics. These include an increase in hybridization affinity toward a complementary RNA (1.5 degrees C per modification) and an increase in resistance toward both 3'-exonuclease and intracellular nucleases. These improvements result in a substantial increase in oligonucleotide potency (>20-fold after 72 h). The most active compound identified was used to examine the role played by protein kinase C-alpha in mediating the phorbol ester-induced changes in c-fos, c-jun, and junB expression in A549 lung epithelial cells. Depletion of protein kinase C-alpha protein expression by this oligonucleotide lead to a reduction in c-jun expression but not c-fos or junB. These results demonstrate that 2'-O-(2-methoxy)ethyl-modified antisense oligonucleotides are 1) effective inhibitors of protein kinase C-alpha expression, and 2) represent a class of antisense oligonucleotide which are much more effective inhibitors of gene expression than the widely used phosphorothioate antisense oligodeoxynucleotides.
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